Protein purification methods to reduce acidic species

ABSTRACT

The instant invention relates to the field of protein production and purification, and in particular to compositions and processes for controlling the amount of charge variants, aggregates, and fragments of a protein of interest, as well as host cell proteins, present in purified preparations by applying particular chromatography conditions during such protein purification.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/636,493, filed on Apr. 20, 2012, the disclosure of which isincorporated by reference herein in its entirety.

1. INTRODUCTION

The instant invention relates to the field of protein production andpurification, and in particular to compositions and processes forcontrolling the amount of product-related substances (e.g., productcharge variants, aggregates, and fragments) and/or process-relatedimpurities (e.g., host cell proteins and media components) present inpurified preparations by applying particular chromatography conditionsduring such protein purification.

2. BACKGROUND OF THE INVENTION

The production of proteins for biopharmaceutical applications typicallyinvolves the use of cell cultures that are known to produce proteinsexhibiting varying levels of heterogeneity. Such heterogeneity includes,but is not limited to, the presence of product-related species, such ascharged species heterogeneity, consisting of acidic species and basicspecies. In monoclonal antibody (mAb) preparations, such acidic speciesheterogeneities can be detected by various methods, such as WCX-10 HPLC(a weak cation exchange chromatography) or IEF (isoelectric focusing).In certain embodiments, the acidic species identified using suchtechniques comprise a range of product-related substances such asantibody product fragments (e.g., Fe and Fab fragments), and/orpost-translation modifications of the antibody product, such as,deamidated and/or glycoslyated antibodies. For example, in a sample ofthe human IgG antibody adalimumab, WCX-10 analysis measured the presenceof acidic species that can be divided, based on residence time, into twogroups: acidic region 1 (AR1) and acidic region 2 (AR2). Because oftheir similar chemical characteristics to the antibody molecules ofinterest, reduction of acidic species is a particular challenge inmonoclonal antibody purification.

There remains a need in the art for high-efficiency methods of purifyingproteins of interest, e.g., antibodies, away from product-relatedsubstances and process-related impurities at relatively low cost.Reduction of such substances and/or impurities is particularlyadvantageous in the context of commercially produced recombinantbio-therapeutics as such substances and/or impurities have the potentialto impact numerous product characteristics, including, but not limitedto, product stability, product safety and product efficacy.

3. SUMMARY OF THE INVENTION

In certain embodiments, the present invention is directed toprocess-related impurity-reduced and/or product-relatedsubstance-modulated preparations of a protein of interest. In certainembodiments, the process-related impurities include, but are not limitedto host cell proteins (HCPs), host nucleic acids, chromatographicmaterials, and media components. In certain embodiments, theproduct-related substances include, but are not limited to chargevariants, such as acidic species and basic species. In certainembodiments, such acidic species correspond to heterogeneity in thedistribution of protein fragments (e.g., Fc and Fab fragments ofantibodies), and/or post-translation modifications of the proteins, suchas, deamidated and/or glycoslyated proteins, in the population ofproteins, and such heterogeneity particularly of interest when it arisesin the context of recombinant protein production. Further, the presentinvention is directed toward pharmaceutical compositions comprising oneor more proteins purified by a method described herein. In anotheraspect, such compositions further comprise one or more pharmaceuticalagents.

In certain embodiments, the present invention is directed to a methodfor preparing a process-related impurity-reduced and/or product-relatedsubstance-modulated composition comprising a protein of interest whereina chromatographic separation is performed to identify the particularconditions, e.g., salt concentration, pH, temperature, load amount andconditions, and washing conditions, sufficient to elicit the desiredfractionation profile, e.g., fractionation of product-relatedsubstances, such as acidic species and lysine variants, of a samplecomprising the protein of interest and at least one process-relatedimpurity and/or at least one product-related substance. In certainembodiments, the method will further comprise pooling of the resultingfractions comprising the desired process-related impurity-reduced and/orproduct-related substance-modulated composition comprising a protein ofinterest.

In certain embodiments, the present invention is directed to methods forisolating and purifying a protein, for example, an antibody, or anantigen-binding portion thereof, from a sample in order to exert controlover the presence of process-related impurities and/or product-relatedsubstances.

In certain embodiments, the methods of purifying a protein, such as anantibody or antigen-binding portion thereof, from a sample, as describedherein, reduces the amount of acidic species present in the resultingcomposition. In certain embodiments, the resulting composition issubstantially free of acidic species. In certain embodiments, theresulting composition is substantially free of one or more acidicsub-species, for example, with regard to the purification of Adalimumab,the composition is substantially free of AR1 and/or AR2. In certainembodiments, the methods described herein reduce the amount of host cellproteins (“HCPs”) present in the resulting composition. In certainembodiments, the resulting composition is substantially free of HCPs. Inone aspect, the sample mixture to be purified comprises a partiallypurified cell line harvest wherein the cell line is employed to producespecific proteins of the present invention. In a particular aspect, thesample mixture is prepared from a cell line used to produce anti-TNF-aantibodies.

In certain aspects, the invention is directed to methods of proteinpurification employing chromatography, preferably chromatography thatutilizes a multimodal (also known as “mixed mode” or “MM”) media.

In certain embodiments, the multimodal media comprises functional groupswhich exhibit anion exchange and/or hydrophobic interactions. In certainembodiments, the multimodal media comprises a cross-linked agarose witha ligand, for example, N-Benzyl-N-methyl ethanol amine, that exhibitsionic interactions, hydrogen bonding and hydrophobic interactions. Incertain embodiments, the cross-linked agarose with a ligand(N-Benzyl-N-methyl ethanol amine) has the following structure:

In certain embodiments, the multimodal media comprises a cross-linkedcellulose exhibiting porosity. In certain embodiments, the cross-linkedcellulose is a phenylpropylamine having the following structure:

In certain embodiments, the cross-linked cellulose is a hexylaminehaving the following structure:

—CH₂—(CH₂)₄—CH₃.

In certain embodiments of the present invention, a sample comprising theprotein of interest, such as an antibody or antigen-binding portionthereof, is subjected to chromatography that utilizes a multimodalmedia, wherein the sample is subjected to a pH adjustment duringloading. In one aspect, the pH is adjusted to a basic pH, or an increasein pH. An example of a suitable pH is between about a pH of 7 and 8.2,preferably a pH of between about 7.5 and 8.2. In certain embodiments theselection of appropriate pH will be based on the characteristics of theantibody and/or acidic species of interest. In certain embodiments, thepH will be selected to be about 0 to 3 units lower than the pI of theprotein. In certain embodiments, it is in the range of 1 to 2 unitslower. In certain embodiments, it is in the range of 1 to 1.5 unitslower.

Certain embodiments of the present invention involve subjecting a samplemixture comprising a protein of interest, such as an antibody orantigen-binding portion thereof, to chromatography that utilizes amultimodal media, wherein the sample is subjected to a conductivityadjustment during loading. In one aspect, the conductivity is adjustedto between about 1 and 86 mS/cm, preferably between about 2 and 14mS/cm. In certain embodiments, alternative ranges of conductivity areemployed, which would be based on the characteristics of the antibodyand/or acidic species of interest.

Certain embodiments of the present invention involve subjecting a samplecomprising a protein of interest, such as an antibody or antigen-bindingportion thereof, to chromatography that utilizes a multimodal media,wherein the sample is subjected to an adjustment in the amount ofprotein load used in the multimodal chromatography. In one aspect, thetotal protein load to the column is of between about 5 and 1000 g/L, orbetween about 50 and 500 g/L, between about 75 and 300 g/L, or betweenabout 100 and 250 g/L. In certain embodiments, the protein concentrationof the load protein mixture is adjusted to a protein concentration ofthe material loaded to the column of about 0.5 and 50 g/L, or betweenabout 1 and 20 g/L.

In certain embodiments, the methods of the present invention involvessubjecting a sample comprising a protein of interest, such as anantibody or antigen-binding portion thereof, to chromatography thatutilizes an anion exchange (AEX) adsorbent material and an aqueous saltsolution under loading conditions that permit both the protein ofinterest and non-target proteins to bind to the AEX adsorbent andcollecting any unbound material with reduced levels of acidic species(and optionally reduced levels of one or more product relatedimpurities/substances or process related impurities) and subsequentlywing the adsorbent with awash buffer comprising the same, orsubstantially similar, aqueous salt solution used in the loading sampleand collecting the effluent containing reduced levels of acidic species(and optionally reduced levels of one or more product relatedimpurities/substances or process related impurities). In certainembodiments, the salt concentration is between 0.5 mM and 50 mM, or 2 mMand 40 mM, or 5 mM and 20 mM, depending on the salt type and AEXadsorbent being used. In certain embodiments, the concentration of theanionic and/or cationic agent in aqueous salt solution is increased ordecreased to achieve a pH of between about 5 and 10, or between about 7and 9. In certain embodiments, the aqueous salt solution comprises ananionic agent at a concentration of about 5 mM, 10 mM or 18.5 mM, and anamount of a cationic agent sufficient to achieve a pH of 8.8 or 9.0.

In certain embodiments of the present invention involve subjecting asample comprising the putative protein of interest, such as an antibodyor antigen-binding portion thereof, to chromatography that utilizes aAEX, wherein the sample is subjected to an adjustment in the amount ofprotein concentration and load. In one aspect, the total protein load tothe column of between about 50 and 500 g/L, or between about 75 and 350g/L, or between about 200 and 300 g/L. In certain embodiments, theprotein concentration of the load protein mixture is adjusted to aprotein concentration of the material loaded to the column of about 0.5and 50 g/L, between about 1 and 20 g/L, or between 3 and 10 g/L.

In certain embodiments, the methods of the present invention involvessubjecting a sample comprising a putative protein of interest, such asan antibody or antigen-binding portion thereof, to chromatography thatutilizes a cation exchange (CEX) adsorbent material and an aqueoussolution under loading conditions that permit both the protein ofinterest and non-target proteins to bind to the CEX adsorbent, whereinacidic species and non-target proteins are washed from the CEX adsorbentmaterial using a wash buffer comprising the same, or substantiallysimilar, aqueous solution as the loading buffer, and wherein the boundprotein of interest is subsequently recovered with an elution bufferhaving a higher conductivity than the loading buffer.

In certain embodiments of the present invention involve subjecting asample comprising the protein of interest, such as an antibody orantigen-binding portion thereof, to chromatography that utilizes a CEX,wherein the aqueous solution for loading and wash is a combination of pHand ionic concentration to allow the removal the acidic species in theunbound wash fractions. The pH employed in certain of such embodimentsis adjusted such that it is below the pI of the protein of interest.

In certain embodiments of the present invention involve subjecting asample comprising the putative protein of interest, such as an antibodyor antigen-binding portion thereof, to chromatography that utilizes aCEX, wherein the sample is subjected to an adjustment in the amount ofprotein concentration and load. In one aspect, the total protein load tothe column of between about 5 and 150 g/L, or between about 10 and 100g/L, between about 20 and 80 g/L, or between about 30 and 50 g/L. Incertain embodiments, the protein concentration of the load proteinmixture is adjusted to a protein concentration of the material loaded tothe column of about 0.5 and 50 g/L, or between about 1 and 20 g/L.

In certain embodiments, control over the amount of acidic species in theprotein compositions described herein is exerted by employing one ormore of the foregoing methods during the production and purification ofthe desired proteins, such as antibodies or antigen-binding portionsthereof, described herein.

In certain embodiments, the sample is subject to a first chromatographicstep prior to the multimodal/AEX or CEX media chromatography describedabove. The sample in each case is prepared appropriately to achieve thetarget pH and ion concentration prior to separation on the differentmodes of chromatography. Such prior chromatographic steps include ionexchange and/or affinity chromatography. Non-limiting examples ofchromatographic supports for use in the first chromatographic stepinclude, but are not limited to, affinity chromatographic resins, suchas, but not limited to, Protein A resin and Protein G resin, or otheraffinity supports such as those comprising the antigen against which anantibody of interest was raised, as well as affinity supports comprisingan Fc binding protein. In one aspect, where the protein of interest isan antibody, a sample is loaded on a protein A affinity columnchromatography and eluted with a buffer system containing buffercomponents to be used in the multimodal media chromatography. During lowpH viral inactivation, the pH is adjusted to pH 3.5 to 3.7 with acid(e.g., the same as the acidic component of the multimodal mediachromatography equilibration buffer system) and held for about 30 to 90minutes. The material is then neutralized with base) to the designed pH.In certain embodiments, the buffer is a Tris/acetate buffer system. Incertain embodiments, the buffer is a trolamine/NaCl buffer. The materialis then clarified with filters. The eluate can be monitored usingtechniques well known to those skilled in the art. For example, theabsorbance at OD₂₈₀ can be followed. The eluated fraction(s) of interestcan then be prepared for further processing with multimodalchromatography, AEX or CEX media chromatography.

The purity of the proteins of interest, including but not limitedprocess-related impurities and product-related substances, in the sampleresulting from practice of the chromatographic strategies describedherein can be analyzed using methods well known to those skilled in theart, e.g., weak cation exchange chromatography (WCX), capillaryisoelectric focusing (cIEF), size-exclusion chromatography, Poros™ AHPLC Assay, HCP ELISA, Protein A ELISA, and western blot analysis.

4. BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 depicts a process chromatogram of pH gradient elution in thecontext of AEX chromatography.

FIG. 2 depicts a process chromatogram of a linear gradient elution byincreasing anion concentration in the context of AEX chromatography.

FIG. 3 depicts a process chromatogram of fractionation of 300 g/L loadand wash in the context of AEX chromatography.

FIG. 4 depicts the effect of pH on AR reduction in the context of AEXchromatography.

FIG. 5 depicts a process chromatogram at different salt (cation)concentrations in the context of CEX chromatography.

FIG. 6 depicts recovery versus AR reduction in the context of CEXpurification of adalimumab.

FIG. 7 depicts the WCX-10 profile of glycated load material and CEXEluate.

FIG. 8 depicts the WCX 10 profile of MGO modified load material andeluate from CEX column employing Toyo Pearl MX TRP 650M resin.

FIG. 9 depicts the change in Lysine distribution during CEXchromatography, highlighting the effect of Tris concentration.

FIG. 10 depicts the effect of pH and conductivity on Adalimumab ARreduction and recovery yield in the context of MM chromatography.

FIG. 11 depicts the AR reduction achieved with the corresponding proteinrecovery in the context of MM chromatography.

FIG. 12 depicts the total Adalimumab Protein concentration levels and ARlevels during Flow Through and Wash.

FIG. 13 depicts the total mAb B Protein concentration levels and ARlevels during Flow Through and Wash in the context of MM chromatography.

FIG. 14 depicts the total mAb C Protein concentration levels and ARlevels during Flow Through and Wash in the context of MM chromatography.

FIG. 15 depicts the Cumulative % AR breakthrough of mAb C on differentMM resins.

FIG. 16 depicts the impact of pH-pI and Conductivity on D2E7(Adalimumab) AR Reduction in the context of MM chromatography.

FIG. 17 depicts the impact of pH-pI and Conductivity on mAb B ARReduction in the context of MM chromatography.

FIG. 18 depicts the impact and trend of pH-pI on mAb C AR reduction withmultiple resins in the context of MM chromatography.

FIG. 19 depicts the effect of pH and Conductivity on AR reduction andYield in the context of MM chromatography.

FIG. 20 depicts AR reduction and Protein recovery vs. pH in the contextof MM chromatography.

FIG. 21 depicts the effect of pH, conductivity and protein load amounton AR reduction and Yield.

FIG. 22 depicts the effect of pH, conductivity and protein load amounton AR reduction and Yield.

FIG. 23 depicts the AR Growth at 25° C. of low and high AR containingsamples.

FIG. 24 depicts the effect of AEX adsorbent pKa for mAb B with severaldifferent AEX adsorbents, with different pKa values, run at with anacetate/Tris buffer at pH 9.1.

5. DETAILED DESCRIPTION OF THE INVENTION

The instant invention relates to the field of protein purification. Inparticular, the instant invention relates to compositions and processesfor controlling the amount of product-related substances (e.g., productcharge variants, aggregates, and fragments) and/or process-relatedimpurities (e.g., host cell proteins and media components) present inpurified preparations of a protein of interest. In certain embodiments,the methods described herein involve the purification of a protein, suchas, but not limited to an antibody or antigen-binding portion thereof,by multimodal chromatography, wherein the multimodal (MM) mediacomprises both ion exchange and hydrophobic interaction functionalgroups and an aqueous salt solution, wherein the same or substantiallythe same aqueous salt solution is used as a loading buffer and a washbuffer using which the said protein of interested is collected with, inthe column effluent.

In certain embodiments, the methods described herein involve thepurification of a protein, such as, but not limited to an antibody orantigen-binding portion thereof, by chromatography comprising an anionexchange (AEX) adsorbent material and an aqueous salt solution, whereinthe same or substantially the same aqueous salt solution is used as aloading buffer and a wash buffer using which the said protein ofinterested is collected with, in the column effluent. In certainembodiments, the methods described herein involve the purification of aprotein, such as, but not limited to an antibody or antigen-bindingportion thereof, by chromatography comprising a cation exchange (CEX)adsorbent material and an aqueous salt solution, wherein the same orsubstantially the same aqueous salt solution is used as a loading bufferand a wash buffer, wherein the wash buffer removes acidic species andnon-target proteins bound to the CEX adsorbent material, and wherein thetarget protein bound to the CEX adsorbent material is eluted with abuffer having a higher conductivity and/or pH than the loading/washbuffer. In certain embodiments, the present invention is directed towardpharmaceutical compositions comprising one or more proteins, such as,but not limited to an antibody or antigen-binding portion thereof,purified by a method described herein.

For clarity and not by way of limitation, this detailed description isdivided into the following sub-portions:

-   -   5.1. Definitions;    -   5.2. Antibody Generation;    -   5.3. Antibody Production;    -   5.4. Antibody Purification;    -   5.5. Methods of Assaying Sample Purity;    -   5.6. Further Modifications; and    -   5.7. Pharmaceutical Compositions

5.1. Definitions

In order that the present invention may be more readily understood,certain terms are first defined.

The term “product”, as used herein refers to a protein of interest,which may be present in the context of a sample comprising one or moreprocess-related impurities and/or product-related substances. In certainembodiments, the product, i.e., the protein of interest is an antibodyor antigen binding fragment thereof.

The term “antibody” includes an immunoglobulin molecule comprised offour polypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as HCVR or VH) and aheavy chain constant region (CH). The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The term “antibody”, as used herein, also includesalternative antibody and antibody-like structures, such as, but notlimited to, dual variable domain antibodies (DVD-Ig).

The term “antigen-binding portion” of an antibody (or “antibodyportion”) includes fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., hIL-12, hTNFα, or hIL-18). It hasbeen shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment comprisingthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment comprising the VH and CH1 domains;(iv) a Fv fragment comprising the VL and VH domains of a single arm ofan antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546,the entire teaching of which is incorporated herein by reference), whichcomprises a VH domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see, e.g., Birdet al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883, the entire teachings of which areincorporated herein by reference). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123, theentire teachings of which are incorporated herein by reference). Stillfurther, an antibody may be part of a larger immunoadhesion molecule,formed by covalent or non-covalent association of the antibody with oneor more other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101, the entire teaching of which isincorporated herein by reference) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058, the entire teaching of which is incorporatedherein by reference). Antibody portions, such as Fab and F(ab′)2fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein. In one aspect, the antigen binding portions arecomplete domains or pairs of complete domains.

The terms “Kabat numbering” “Kabat definitions” and “Kabat labeling” areused interchangeably herein. These terms, which are recognized in theart, refer to a system of numbering amino acid residues which are morevariable (i.e., hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, the entire teachings ofwhich are incorporated herein by reference). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

The term “human antibody” includes antibodies having variable andconstant regions corresponding to human germline immunoglobulinsequences as described by Kabat et al. (See Kabat, et al. (1991)Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), e.g., in the CDRs and in particular CDR3. Themutations can be introduced using the “selective mutagenesis approach.”The human antibody can have at least one position replaced with an aminoacid residue, e.g., an activity enhancing amino acid residue which isnot encoded by the human germline immunoglobulin sequence. The humanantibody can have up to twenty positions replaced with amino acidresidues which are not part of the human germline immunoglobulinsequence. In other embodiments, up to ten, up to five, up to three or upto two positions are replaced. In one embodiment, these replacements arewithin the CDR regions. However, the term “human antibody”, as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences.

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes (see,e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295, theentire teaching of which is incorporated herein by reference) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences(see, Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.In certain embodiments, however, such recombinant antibodies are theresult of selective mutagenesis approach or back-mutation or both.

An “isolated antibody” includes an antibody that is substantially freeof other antibodies having different antigenic specificities. Moreover,an isolated antibody may be substantially free of other cellularmaterial and/or chemicals.

The term “Koff”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “Kd”, as used herein, is intended to refer to the dissociationconstant of a particular antibody-antigen interaction.

The phrase “nucleic acid molecule” includes DNA molecules and RNAmolecules. A nucleic acid molecule may be single-stranded ordouble-stranded, but in one aspect is double-stranded DNA.

The phrase “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3), e.g. an antibody having a weak binding capacity for a Protein Aresin. The phrase “isolated nucleic acid molecule” is also intended toinclude sequences encoding bivalent, bispecific antibodies, such asdiabodies in which VH and VL regions contain no other sequences otherthan the sequences of the diabody.

The phrase “recombinant host cell” (or simply “host cell”) includes acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “modifying”, as used herein, is intended to refer to changingone or more amino acids in the antibodies or antigen-binding portionsthereof. The change can be produced by adding, substituting or deletingan amino acid at one or more positions. The change can be produced usingknown techniques, such as PCR mutagenesis.

The term “about”, as used herein, is intended to refer to ranges ofapproximately 10-20% greater than or less than the referenced value. Incertain circumstances, one of skill in the art will recognize that, dueto the nature of the referenced value, the term “about” can mean more orless than a 10-20% deviation from that value.

The term “preparative scale”, as used herein, refers to a scale ofpurification operation that can be readily scaled-up and implemented atlarge scale manufacturing while still providing desired separation. Forinstance, one skilled in the field may develop a process using, e.g., a0.5 cm (i.d.)×20 cm (L) column in the lab, and transfer it to largescale production using, e.g., a 30 cm (i.d.)×20 cm (L) column packedwith the same resin and operated with the same set of buffers, samelinear flow rates (or residence times) and buffer volumes. Inpreparative scale separation, column bed height is typically ≦about 30cm and column pressure drop ≦about 5 bar.

The term “aggregates” used herein means agglomeration or oligomerizationof two or more individual molecules, including but not limiting to,protein dimers, trimers, tetramers, oligomers and other high molecularweight species. Protein aggregates can be soluble or insoluble.

The term “fragments” used herein refers to any truncated protein speciesfrom the target molecule due to dissociation of peptide chain, enzymaticand/or chemical modifications. For instance, antibody fragments include,but not limited to, Fab, F(ab′)₂, Fv, scFv, Fd, dAb, or othercompositions that contain a portion of the antibody molecule.

The term “charge variants”, as used herein, refers to the fullcomplement of product variants including, but not limited to acidicspecies and basic species (e.g., Lys variants). In certain embodiments,such variants can include product aggregates and/or product fragments,to the extent that such aggregation and/or fragmentation results in aproduct charge variation.

As used herein, the term “lysine variant heterogeneity” refers to acharacteristic of a population of proteins wherein the populationconsists of proteins of substantially identical amino acid sequence, butwhere the population exhibits variation in the presence or absence ofC-terminal lysine residues. Although such lysine variant heterogeneitycan be observed under general cell culture conditions, the use ofparticular cell culture conditions, as detailed below, can increase ordecrease the distribution or amount of lysine variant heterogeneity.

In certain embodiments, the protein is an antibody, and the distributionof lysine variant heterogeneity comprises a distribution of the lysinevariants Lys 0, Lys 1 and Lys 2, wherein the Lys 0 lysine variantcomprises an antibody with heavy chains that do not comprise aC-terminal lysine, wherein the Lys 1 lysine variant comprises anantibody with one heavy chain that comprises a C-terminal lysine, andwherein the Lys 2 lysine variant comprises an antibody wherein bothheavy chains comprise a C-terminal lysine.

In certain embodiments, C-terminal lysine variants are associated withcharge heterogeneities present in protein preparations, for example,monoclonal antibody (mAb) preparations, produced through a cell cultureprocess. These heterogeneities can be detected by various methods, suchas, for example, WCX-10 HPLC (a weak cation exchange chromatography), orIEF (isoelectric focusing).

In certain embodiments, the heterogeneity arises from subspecies ofprotein differing by the presence or absence of C-terminal lysines. Forexample, the population of proteins may comprise more than onesubspecies of lysine variant. In one non-limiting example, the lysinevariants may comprise at least two of Lys 0, Lys 1 and Lys 2 lysinevariants which can be detected by weak cation exchange chromatography ofthe expression product of a host cell expressing Adalimumab.

In certain embodiments, the heterogeneity arises from the size ofsubpopulations having different C-terminal lysine profiles. For example,the population of proteins may comprise more than one subspecies ofC-terminal lysine variant, and each of the variants may be present indifferent amounts. In one non-limiting example, the C-terminal lysinevariants may be at least two of the Lys 0, Lys 1 and Lys 2 lysinevariants detected by weak cation exchange chromatography of theexpression product of a host cell expressing Adalimumab. In certainembodiments, Lys 0, Lys 1 or Lys 2 subspecies are present in differentamounts.

In certain embodiments, the heterogeneity arises from both a differencein the amount of lysine variants in the population of proteins and thetype of lysine variants present in the population of proteins.

As used herein, the terms “acidic species”, “acidic region” and “acidicspecies heterogeneity” refer to a characteristic of a population ofproteins wherein the population includes a distribution ofproduct-related impurities identifiable by the presence of chargeheterogeneities. For example, in monoclonal antibody (mAb) preparations,such acidic species heterogeneities can be detected by various methods,such as, for example, WCX-10 HPLC (a weak cation exchangechromatography), or IEF (isoelectric focusing). In certain embodiments,the acidic species identified using such techniques comprise a mixtureof product-related impurities containing antibody product fragments(e.g., Fc and Fab fragments), and/or post-translation modifications ofthe antibody product, such as, deamidated and/or glycoslyatedantibodies.

In certain embodiments, the acidic species heterogeneity comprises adifference in the type of acidic species present in the population ofproteins. For example, the population of proteins may comprise more thanone acidic species variant.

In certain embodiments, the heterogeneity of the distribution of acidicspecies comprises a difference in the amount of acidic species in thepopulation of proteins. For example, the population of proteins maycomprise more than one acidic species variant, and each of the variantsmay be present in different amounts.

5.2. Antibody Generation

The term “antibody” as used in this section refers to an intact antibodyor an antigen binding fragment thereof.

The antibodies of the present disclosure can be generated by a varietyof techniques, including immunization of an animal with the antigen ofinterest followed by conventional monoclonal antibody methodologiese.g., the standard somatic cell hybridization technique of Kohler andMilstein (1975) Nature 256: 495. Although somatic cell hybridizationprocedures are preferred, in principle, other techniques for producingmonoclonal antibody can be employed e.g., viral or oncogenictransformation of B lymphocytes.

In certain embodiments, the animal system for preparing hybridomas isthe murine system. Hybridoma production is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

An antibody can be, in certain embodiments, a human, a chimeric, or ahumanized antibody. Humanized antibodies of the present disclosure canbe prepared based on the sequence of a non-human monoclonal antibodyprepared as described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the non-human hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,murine CDR regions can be inserted into a human framework using methodsknown in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen etal.).

Human monoclonal antibodies can be generated using transgenic ortranschromosomic mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice referred to herein as the HuMAb Mouse® (Medarex, Inc.), KMMouse® (Medarex, Inc.), and XenoMouse® (Amgen).

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseantibodies of the disclosure. For example, mice carrying both a humanheavy chain transchromosome and a human light chain tranchromosome,referred to as “TC mice” can be used; such mice are described inTomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727.Furthermore, cows carrying human heavy and light chain transchromosomeshave been described in the art (e.g., Kuroiwa et al. (2002) NatureBiotechnology 20:889-894 and PCT application No. WO 2002/092812) and canbe used to raise the antibodies of this disclosure.

In certain embodiments, the antibodies of this disclosure arerecombinant human antibodies, which can be isolated by screening of arecombinant combinatorial antibody library, e.g., a scFv phage displaylibrary, prepared using human VL and VH cDNAs prepared from mRNA derivedfrom human lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. In addition to commercially availablekits for generating phage display libraries (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612, the entireteachings of which are incorporated herein), examples of methods andreagents particularly amenable for use in generating and screeningantibody display libraries can be found in, e.g., Ladner et al. U.S.Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Doweret al. PCT Publication No. WO 91/17271; Winter et al. PCT PublicationNo. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679;Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCTPublication No. WO 92/01047; Garrard et al. PCT Publication No. WO92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982; the entire teachings of whichare incorporated herein.

Human monoclonal antibodies of this disclosure can also be preparedusing SCID mice into which human immune cells have been reconstitutedsuch that a human antibody response can be generated upon immunization.Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

The antibodies or antigen-binding portions thereof, of this disclosurecan be altered wherein the constant region of the antibody is modifiedto reduce at least one constant region-mediated biological effectorfunction relative to an unmodified antibody. To modify an antibody ofthe invention such that it exhibits reduced binding to the Fe receptor,the immunoglobulin constant region segment of the antibody can bemutated at particular regions necessary for Fc receptor (FcR)interactions (see, e.g., Canfield and Morrison (1991) J. Exp. Med.173:1483-1491; and Lund et al. (1991) J. of Immunol. 147:2657-2662, theentire teachings of which are incorporated herein). Reduction in FcRbinding ability of the antibody may also reduce other effector functionswhich rely on FcR interactions, such as opsonization and phagocytosisand antigen-dependent cellular cytotoxicity.

5.3. Antibody Production

To express an antibody of the invention, DNAs encoding partial orfull-length light and heavy chains are inserted into one or moreexpression vector such that the genes are operatively linked totranscriptional and translational control sequences. (See, e.g., U.S.Pat. No. 6,914,128, the entire teaching of which is incorporated hereinby reference.) In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into a separate vector or, more typically, bothgenes are inserted into the same expression vector. The antibody genesare inserted into an expression vector by standard methods (e.g.,ligation of complementary restriction sites on the antibody genefragment and vector, or blunt end ligation if no restriction sites arepresent). Prior to insertion of the antibody or antibody-related lightor heavy chain sequences, the expression vector may already carryantibody constant region sequences. Additionally or alternatively, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, a recombinant expression vectorof the invention can carry one or more regulatory sequence that controlsthe expression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, e.g., in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990), the entire teaching of which is incorporatedherein by reference. It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, see,e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al., the entireteachings of which are incorporated herein by reference.

In addition to the antibody chain genes and regulatory sequences, arecombinant expression vector of the invention may carry one or moreadditional sequences, such as a sequence that regulates replication ofthe vector in host cells (e.g., origins of replication) and/or aselectable marker gene. The selectable marker gene facilitates selectionof host cells into which the vector has been introduced (see e.g., U.S.Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al., theentire teachings of which are incorporated herein by reference). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Suitable selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr-hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

An antibody of the invention can be prepared by recombinant expressionof immunoglobulin light and heavy chain genes in a host cell. To expressan antibody recombinantly, a host cell is transfected with one or morerecombinant expression vectors carrying DNA fragments encoding theimmunoglobulin light and heavy chains of the antibody such that thelight and heavy chains are expressed in the host cell and secreted intothe medium in which the host cells are cultured, from which medium theantibodies can be recovered. Standard recombinant DNA methodologies areused to obtain antibody heavy and light chain genes, incorporate thesegenes into recombinant expression vectors and introduce the vectors intohost cells, such as those described in Sambrook, Fritsch and Maniatis(eds), Molecular Cloning; A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., (1989), Ausubel et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.Nos. 4,816,397 & 6,914,128, the entire teachings of which areincorporated herein.

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is (are) transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it istheoretically possible to express the antibodies of the invention ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, such as mammalian host cells, is suitable because sucheukaryotic cells, and in particular mammalian cells, are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibodygenes has been reported to be ineffective for production of high yieldsof active antibody (Boss and Wood (1985) Immunology Today 6:12-13, theentire teaching of which is incorporated herein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, e.g., Enterobacteriaceae suchas Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratia marcescans, and Shigella, as well as Bacilli such as B.subtilis and B. lichenifonnis (e.g., B. licheniformis 41P disclosed inDD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa,and Streptomyces. One suitable E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424),K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodies arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

Suitable mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) PNAS USA77:4216-4220, used with a DHFR selectable marker, e.g., as described inKaufman and Sharp (1982) Mol. Biol. 159:601-621, the entire teachings ofwhich are incorporated herein by reference), NSO myeloma cells, COScells and SP2 cells. When recombinant expression vectors encodingantibody genes are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or secretionof the antibody into the culture medium in which the host cells aregrown. Other examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2), the entire teachings of which are incorporated herein byreference.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce an antibody may be cultured in a varietyof media. Commercially available media such as Ham's F10™ (Sigma),Minimal Essential Medium™ ((MEM), (Sigma), RPMI-1640 (Sigma), andDulbecco's Modified Eagle's Medium™ ((DMEM), Sigma) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz, 58:44 (1979), Barnes et al., Anal. Biochem. 102:255(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may beused as culture media for the host cells, the entire teachings of whichare incorporated herein by reference. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas gentamycin drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It is understood thatvariations on the above procedure are within the scope of the presentinvention. For example, in certain embodiments it may be desirable totransfect a host cell with DNA encoding either the light chain or theheavy chain (but not both) of an antibody of this invention. RecombinantDNA technology may also be used to remove some or the entire DNAencoding either or both of the light and heavy chains that is notnecessary for binding to the antigen to which the putative antibody ofinterest binds. The molecules expressed from such truncated DNAmolecules are also encompassed by the antibodies of the invention. Inaddition, bifunctional antibodies may be produced in which one heavy andone light chain are an antibody of the invention and the other heavy andlight chain are specific for an antigen other than the one to which theputative antibody of interest binds, depending on the specificity of theantibody of the invention, by crosslinking an antibody of the inventionto a second antibody by standard chemical crosslinking methods.

In a suitable system for recombinant expression of an antibody of theinvention, a recombinant expression vector encoding both the antibodyheavy chain and the antibody light chain is introduced into dhfr-CHOcells by calcium phosphate-mediated transfection. Within the recombinantexpression vector, the antibody heavy and light chain genes are eachoperatively linked to CMV enhancer/AdMLP promoter regulatory elements todrive high levels of transcription of the genes. The recombinantexpression vector also carries a DHFR gene, which allows for selectionof CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the antibody heavy andlight chains and intact antibody is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. In one aspect, if the antibody is produced intracellularly, as afirst step, the particulate debris, either host cells or lysed cells(e.g., resulting from homogenization), can be removed, e.g., bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems can be firstconcentrated using a commercially available protein concentrationfilter, e.g., an Amicon™ or Millipore Pellicon™ ultrafiltration unit.

Prior to the process of the invention, procedures for purification ofantibodies from cell debris initially depend on the site of expressionof the antibody. Some antibodies can be secreted directly from the cellinto the surrounding growth media; others are made intraceilularly. Forthe latter antibodies, the first step of a purification processtypically involves: lysis of the cell, which can be done by a variety ofmethods, including mechanical shear, osmotic shock, or enzymatictreatments. Such disruption releases the entire contents of the cellinto the homogenate, and in addition produces subcellular fragments thatare difficult to remove due to their small size. These are generallyremoved by differential centrifugation or by filtration. Where theantibody is secreted, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit. Where the antibody is secreted into the medium,the recombinant host cells can also be separated from the cell culturemedium, e.g., by tangential flow filtration. Antibodies can be furtherrecovered from the culture medium using the antibody purificationmethods of the invention.

5.4. Antibody Purification

5.4.1. Antibody Purification Generally

In certain embodiments, the invention provides methods and compositionsfor producing a purified or partially purified (e.g., process-relatedimpurity-reduced and/or product-related substance-modulated) proteinpreparation from a mixture comprising a protein of interest, e.g., anantibody, and at least one process-related impurity or product-relatedsubstance. In certain embodiments, the modulation of product-relatedsubstances involves the reduction of certain of such substances, whilein other embodiments, such modulation can be to increase certain of suchsubstances. For example, in certain embodiments, it is desired to matchthe product-related substance profile of a sample to that of a referencesample by the methods described herein.

In certain embodiments, the compositions of the present inventioninclude, but are not limited to, process-related impurity-reduced and/orproduct-related substance-modulated compositions comprising a protein ofinterest. For example, but not by way of limitation, the presentinvention is directed to process-related impurity-reduced and/orproduct-related substance-modulated compositions comprising Adalimumab.Such process-related impurity-reduced and/or product-relatedsubstance-modulated compositions process-related impurity-reduced and/orproduct-related substance-modulated compositions address the need forimproved product characteristics, including, but not limited to, productstability, product safety and product efficacy.

In certain embodiments, the present invention is directed to a methodfor preparing a process-related impurity-reduced and/or product-relatedsubstance-modulated composition comprising a protein of interest whereina chromatographic separation is performed to identify the particularconditions, e.g., salt concentration, pH, temperature, load amount andconditions, and washing conditions, sufficient to elicit the desiredfractionation profile, e.g., fractionation of product-relatedsubstances, such as acidic species and lysing variants, of a samplecomprising the protein of interest and at least one process-relatedimpurity and/or at least one product-related substance. In certainembodiments, the method will further comprise pooling of the resultingfractions comprising the desired process-related impurity-reduced and/orproduct-related substance-modulated composition comprising a protein ofinterest.

In certain embodiments, the purification process of the invention beginsat the separation step when the antibody has been produced usingproduction methods described above and/or by alternative productionmethods conventional in the art. Once a clarified solution or mixturecomprising the protein of interest, e.g., an antibody, has beenobtained, separation of the protein of interest from process-relatedimpurities, such as the other proteins produced by the cell, as well asproduct-related substances, such as charge variants and/or size variants(aggregates and fragments) is performed. In certain non-limitingembodiments, such separation is performed using CEX, AEX, and/or MMchromatography. In certain embodiments, a combination of one or moredifferent purification techniques, including affinity separationstep(s), ion exchange separation step(s), mixed-mode step(s), and/orhydrophobic interaction separation step(s) can also be employed. Suchadditional purification steps separate mixtures of proteins on the basisof their charge, degree of hydrophobicity, and/or size. In one aspect ofthe invention, such additional separation steps are performed usingchromatography, including hydrophobic, anionic or cationic interaction(or a combination thereof). Numerous chromatography resins are availablefor each of these techniques, allowing accurate tailoring of thepurification scheme to the particular protein involved. The essence ofeach of the separation methods is that proteins can either traverse atdifferent rates down a column, achieving a physical separation thatincreases as they pass further down the column, or to adhere selectivelyto the separation medium, being then differentially eluted by differentsolvents. In some cases, the protein of interest is separated fromimpurities and or product-related substances when the impurities and/orproduct-related substances specifically adhere to the column and theprotein of interest does not, i.e., the protein of interest is washedfrom the column, while in other cases the protein of interest willadhere to the column, while the impurities and/or product-relatedsubstances are washed from the column.

5.4.2. Primary Recovery

In certain embodiments, the initial steps of the purification methods ofthe present invention involve the clarification and primary recovery ofantibody from a sample matrix. In certain embodiments, the primaryrecovery will include one or more centrifugation steps to separate theantibody product from the cells and cell debris. Centrifugation of thesample can be run at, for example, but not by way of limitation, 7,000×gto approximately 12,750×g. In the context of large scale purification,such centrifugation can occur on-line with a flow rate set to achieve,for example, but not by way of limitation, a turbidity level of 150 NTUin the resulting supernatant. Such supernatant can then be collected forfurther purification, or in-line filtered through one or more depthfilters for further clarification of the sample.

In certain embodiments, the initial steps of the purification methods ofthe present invention involve the clarification and primary recovery ofantibody from a sample matrix. In certain embodiments, the primaryrecovery will include one or more centrifugation steps to separate theantibody product from the cells and cell debris. Centrifugation of thesample can be run at, for example, but not by way of limitation, 7,000×gto approximately 12,750×g. In the context of large scale purification,such centrifugation can occur on-line with a flow rate set to achieve,for example, but not by way of limitation, a turbidity level of 150 NTUin the resulting supernatant. Such supernatant can then be collected forfurther purification, or in-line filtered through one or more depthfilters for further clarification of the sample,

In certain embodiments, the primary recovery will include the use of oneor more depth filtration steps to clarify the sample matrix and therebyaid in purifying the antibodies of interest in the present invention. Inother embodiments, the primary recovery will include the use of one ormore depth filtration steps post centrifugation to further clarify thesample matrix. Non-limiting examples of depth filters that can be usedin the context of the instant invention include the Millistak+ X0HC,F0HC, D0HC, A1HC, B1HC depth filters (EMD Millipore), Cuno™ model30/60ZA, 60/90 ZA, VR05, VR07, delipid depth filters (3M Corp.). A 0.2μm filter such as Sartorius's 0.45/0.2 μm Sartopore™ bi-layer orMillipore's Express SHR or SHC filter cartridges typically follows thedepth filters.

In certain embodiments, the primary recovery process can also be a pointat which to reduce or inactivate viruses that can be present in thesample matrix. For example, any one or more of a variety of methods ofviral reduction/inactivation can be used during the primary recoveryphase of purification including heat inactivation (pasteurization), pHinactivation, solvent/detergent treatment, UV and γ-ray irradiation andthe addition of certain chemical inactivating agents such asβ-propiolactone or e.g., copper phenanthroline as in U.S. Pat. No.4,534,972. In certain embodiments of the present invention, the samplematrix is exposed to detergent viral inactivation during the primaryrecovery phase. In other embodiments, the sample matrix may be exposedto low pH inactivation during the primary recovery phase.

In those embodiments where viral reduction/inactivation is employed, thesample mixture can be adjusted, as needed, for further purificationsteps. For example, following low pH viral inactivation, the pH of thesample mixture is typically adjusted to a more neutral pH, e.g., fromabout 4.5 to about 8.5, prior to continuing the purification process.Additionally, the mixture may be diluted with water for injection (WFI)to obtain a desired conductivity.

5.4.3. Protein A Affinity Chromatography

In certain embodiments, particularly where the protein of interest is anantibody, the primary recovery sample is subjected to Protein A affinitychromatography to purify the antibody of interest away fromprocess-related impurities, such as HCPs. There are a variety ofcommercial sources for Protein A resin. Suitable resins include, but notlimited to, MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelectXtra, rProtein A Sepharose from GE Healthcare, ProSep HC, ProSep Ultra,and ProSep Ultra Plus from EMD Millipore, MapCapture from LifeTechnologies.

In certain embodiments, the Protein A column can be equilibrated with asuitable buffer prior to sample loading. Following the loading of thecolumn, the column can be washed one or multiple times using a suitablesets of buffers. The Protein A column can then be eluted using anappropriate elution buffer. The eluate can be monitored using techniqueswell known to those skilled in the art. The eluate fractions of interestcan be collected and then prepared for further processing.

The Protein A eluate may subject to a viral inactivation step either bydetergent or low pH, provided this step is not performed prior to theProtein A capture operation. A proper detergent concentration or pH andtime can be selected to obtain desired viral inactivation results. Afterviral inactivation, the Protein A eluate is usually pH and/orconductivity adjusted for subsequent purification steps.

The Protein A eluate may be subjected to filtration through a depthfilter to remove turbidity and/or various impurities from the antibodyof interest prior to additional chromatographic polishing steps.Examples of depth filters include, but not limited to, Millistak+X0HC,F0HC, D0HC, A1HC, and B1HC Pod filters (EMD Millipore), or Zeta Plus30ZA/60ZA, 60ZA/90ZA, delipid, VR07, and VR05 filters (3M). The ProteinA eluate pool may need to be conditioned to proper pH and conductivityto obtain desired impurity removal and product recovery from the depthfiltration step.

5.4.5. Anion Exchange Chromatography

In certain embodiments, the instant invention provides methods forproducing a process-related impurity and/or product-relatedsubstance-reduced protein preparation from a mixture comprising aprotein of interest (i.e., a product) and at least one process-relatedimpurity and/or product-related substance by subjecting the mixture toat least one anion exchange separation step. In certain embodiments, theanion exchange step will occur after the above-described Protein Aaffinity step.

The use of an anionic exchange material versus a cationic exchangematerial, such as those cation exchange materials discussed in detailbelow, is based on the local charges of the protein of interest in agiven solution. Therefore, it is within the scope of this invention toemploy an anionic exchange step prior to the use of a cationic exchangestep, or a cationic exchange step prior to the use of an anionicexchange step. Furthermore, it is within the scope of this invention toemploy only an anionic exchange step, only an cationic exchange step, orany serial combination of the two (including serial combinations of oneor both ion exchange steps with the other chromatographic separationtechnologies described herein).

In performing the separation, the initial protein mixture can becontacted with the anion exchange material by using any of a variety oftechniques, e.g., using a batch purification technique or achromatographic technique.

For example, in the context of batch purification, anion exchangematerial is prepared in, or equilibrated to, the desired startingbuffer. Upon preparation, or equilibration, a slurry of the anionexchange material is obtained. The protein of interest, e.g., antibody,solution is contacted with the slurry to allow for protein adsorption tothe anion exchange material. The solution comprising the process-relatedimpurities and/or product-related substances that do not bind to the AEXmaterial is separated from the slurry, e.g., by allowing the slurry tosettle and removing the supernatant. The slurry can be subjected to oneor more washing steps and/or elution steps.

In the context of chromatographic separation, a chromatographicapparatus, commonly cylindrical in shape, is employed to contain thechromatographic support material (e.g., AEX material) prepared in anappropriate buffer solution. The chromatographic apparatus, ifcylindrical, can have a diameter of about 5 mm to about 2 meters, and aheight of 5 cm to 50 cm, and in certain embodiments, particularly forlarge scale processing, a height of <30 cm is employed. Once thechromatographic material is added to the chromatographic apparatus, asample containing the protein of interest, e.g., an antibody, iscontacted to the chromatographic material to induce the separation. Anyportion of the solution that does not bind to the chromatographicmaterial, e.g., which may comprise, depending on the AEX material beingemployed, the protein of interest, process-related impurities, and/orproduct-related substances, is separated from the chromatographicmaterial by washing the material and collecting fractions from column.The chromatographic material can be subjected to one or more wash steps.If desired, the chromatographic material can then be contacted with asolution designed to desorb any components of the solution that havebound to the chromatographic material.

In certain embodiments, a wash step can be performed in the context ofAEX chromatography using conditions similar to the load conditions oralternatively by decreasing the pH and/or increasing the ionicstrength/conductivity of the wash in a step wise or linear gradientmanner. The resulting flow through and wash fractions can be analyzedand appropriate fractions pooled to achieve the desired reduction incharged variant species. In certain embodiments, the aqueous saltsolution used as both the loading and wash buffer has a pH that at ornear the isoelectric point (pI) of the protein of interest. In certainembodiments the pH is about 0 to 2 units higher or lower than the pI ofthe protein of interest. In certain embodiments, it will be in the rangeof 0 to 0.5 units higher or lower. In certain embodiments, it will be atthe pI of the antibody.

In certain non-limiting embodiments, the anionic agent is selected fromthe group consisting of acetate, formate, or combinations thereof. Incertain non-limiting embodiments, the cationic agent is selected fromthe group consisting of Tris, arginine, or combinations thereof.

A packed anion-exchange chromatography column, anion-exchange membranedevice, anion-exchange monolithic device, or depth filter media can beoperated either in bind-elute mode, flow-through mode, or a hybrid modewherein the product exhibits binding to the chromatographic material,yet can be washed from the column using a buffer that is the same orsubstantially similar to the loading buffer. In the bind-elute mode, thecolumn or the membrane device is first conditioned with a buffer withappropriate ionic strength and pH under conditions where certainproteins will be immobilized on the resin based matrix. For example, incertain embodiments, during the feed load, the protein of interest willbe adsorbed to the resin due to electrostatic attraction. After washingthe column or the membrane device with the equilibration buffer oranother buffer with different pH and/or conductivity, the productrecovery is achieved by increasing the ionic strength (i.e.,conductivity) of the elution buffer to compete with the solute for thecharged sites of the anion exchange matrix. Changing the pH and therebyaltering the charge of the solute is another way to achieve elution ofthe solute. The change in conductivity or pH may be gradual (gradientelution) or stepwise (step elution). In the flow-through mode, thecolumn or the membrane device is operated at selected pH andconductivity such that the protein of interest does not bind to theresin or the membrane while the process-related impurities andproduct-related substances will either be retained on the column or willhave a distinct elution profile as compared to the protein of interest.In the context of this hybrid strategy, process-related impurities andproduct-relates substances will bind to the chromatographic material (orflow through) in a manner distinct from the protein of interest, e.g.,while the protein of interest and certain aggregates and/or fragments ofthe protein of interest may bind the chromatographic material, washesthat preferentially remove the protein of interest can be applied. Thecolumn is then regenerated before next use.

Non-limiting examples of anionic exchange substituents includediethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternaryamine (Q) groups. Additional non-limiting examples include: Poros 50PIand Poros 50HQ, which are a rigid polymeric bead with a backboneconsisting of cross-linked poly[styrene-divinylbenzene]; Capto Q Impresand Capto DEAE, which are a high flow agarose bead; Toyopearl QAE-550,Toyopearl DEAE-650, and Toyopearl GigaCap Q-650, which are a polymericbase bead; Fractogel® EMD TMAE Hicap, which is a synthetic polymericresin with a tentacle ion exchanger; Sartobind STIC® PA nano, which is asalt-tolerant chromatographic membrane with a primary amine ligand;Sartobind Q nano; which is a strong anion exchange chromatographicmembrane; CUNO BioCap; which is a zeta-plus depth filter mediaconstructed from inorganic filter aids, refined cellulose, and an ionexchange resin; and X0HC, which is a depth-filter media constructed frominorganic filter aid, cellulose, and mixed cellulose esters. Thedetailed information is listed in Table 1.

TABLE 1 List of AEX Adsorbent Properties Particle/ AEX Adsorbent VendorMedia Type Ligand Type Pore Size Catalog Number Poros PI Applied ResinWeak ~50 μm 1-2459-11 Poros HQ Biosystems Strong ~50 μm 1-2559-11 CaptoDEAE GE Weak ~90 μm 17-5443-10 CaptoQ Impres Strong ~90 μm 17-5316-10QAE-550 Tosoh Strong ~100 μm 43271 DEAE-650 Weak ~65 μm 43201 GigaCapQ-650 Strong ~75 μm 21854 TMAE HiCap EMD/Millipore Strong ~40-90 μm1.16881.0013 Sartobind STIC ® Sartorius Membrane Weak 3-5 μm92STPA42DN-11-A PA Nano Sartobind Q Nano Strong 3-5 μm 92IEXQ42DN-11CUNO BioCap 25 3M Depth Filter NA NA BC0025L60ZA05A XOHC Millipore NA NAMX0HC23CL3

In certain embodiments, the protein load of the mixture comprisingprotein of interest is adjusted to a total protein load to the column ofbetween about 50 and 500 g/L, or between about 75 and 350 g/L, orbetween about 200 and 300 g/L. In certain embodiments, the proteinconcentration of the load protein mixture is adjusted to a proteinconcentration of the material loaded to the column of about 0.5 and 50g/L, between about 1 and 20 g/L, or between 3 and 10 g/L,

In certain embodiments, additives such as poly ethylene glycol,detergents, amino acids, sugars, chaotropic agents can be added toenhance the performance of the separation, so as to achieve betterrecovery or product quality.

In certain embodiments, including, but not limited to those relating toAdalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR charge variants in the flow through and wash fractions whileenriching for the same in the flow elution fraction, thereby producingprotein preparations with reduced or free of AR variants. In certainembodiments relating to the purification of Adalimumab, the methods ofthe instant invention can be used to selectively remove, significantlyreduce, or essentially remove all of AR1 charge variants in the flowthrough and wash fractions while enriching for the same in the flowelution fraction, thereby producing protein preparations with reduced orfree of AR1 variants. In certain embodiments relating to Adalimumab, themethods of the instant invention can be used to selectively remove,significantly reduce, or essentially remove all of AR2 charge variantsin the flow-through and wash fractions while enriching for the same inthe flow elution fraction, thereby producing protein preparations withreduced or free of AR2 variants.

In certain embodiments, including but not limited to those relating toAdalimiumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofthe methylglyoxal (MGO) variants in the flow through and wash fractionswhile enriching for the same in the elution fraction, thereby producingprotein preparations with reduced or free of MGO variants (for example,see U.S. patent application having the attorney reference no.ABV11886USL1). In certain embodiments, including, but not limited tothose relating to Adalimumab, the methods of the instant invention canbe used to selectively remove, significantly reduce, or essentiallyremove all of the glycated variants (schiff's base and permanentlyglycated forms) in the flow through and wash fractions while enrichingfor the same in the elution fraction, thereby producing proteinpreparations with reduced or free of glycated variants.

In certain embodiments, the loading, pH, conductivity of the AEXchromatography step, as well as elution pH conductivity, can be modifiedto achieve a desired distribution of process-related impurities and/orproduct-relates substances. For example, but not by way of limitation,certain embodiments are directed to the modulation of the lysinedistribution of purified sample of a protein of interest, e.g.,increasing Lys0 and decreasing Lys1 and Lys2. In certain embodiments,the methods of the present invention allow for the preparation ofsamples wherein the amount of Lys0 is decreased, while the amount ofLys1 and/or Lys2 is increased.

In certain embodiments, an AEX chromatographic separation can beperformed and combinations of fractions can be pooled to achieve acombination of desired process-related impurity and/or product-relatessubstance levels, in addition to, or in place of merely modulatingcharge variant concentration.

In certain embodiments, spectroscopy methods such as UV, NIR, FTIR,Fluorescence, Raman may be used to monitor levels of product-relatedcharge variants, aggregates, low molecular weight variants (e.g.,fragments of the protein of interest) in an on-line, at-line or in-linemode, which can then be used to control the level of charge variants,e.g., acidic species, in the pooled material collected from the AEXeffluent. In certain embodiments, specific signals arising from thechemical modification of the proteins such as glycation, MGOmodification, deamidation, glycosylation may be specifically measurableby spectroscopic methods through such in-line, on-line or at-linemethods, enabling realtime or near-real time control of product qualityof the resulting product. In certain embodiments, on-line, at-line orin-line monitoring methods can be used either on the effluent line ofthe chromatography step or in the collection vessel, to enableachievement of the desired product quality/recovery. In certainembodiments, the UV signal can be used as a surrogate to achieve anappropriate product quality/recovery, wherein the UV signal can beprocessed appropriately, including, but not limited to, such processingtechniques as integration, differentiation, moving average, such thatnormal process variability can be addressed and the target productquality can be achieved. In certain embodiments, such measurements canbe combined with in-line dilution methods such that ionconcentration/conductivity of the load/wash can be controlled byfeedback and hence facilitate product quality control.

In certain embodiments, a combination of AEX and CEX and MM methods canbe used to prepare product-related substance-modulated materials,including certain embodiments where one technology is used in acomplementary/supplementary manner with another technology. In certainembodiments, such a combination can be performed such that certainsub-species are removed predominantly by one technology, such that thecombination provides the desired final composition/product quality. Incertain embodiments, such combinations include the use of additionalintervening chromatography, filtration, pH adjustment, UF/DF steps so asto achieve the desired product quality, ion concentration, and/or viralreduction.

5.4.6. Cation Exchange Chromatography

In certain embodiments, the instant invention provides methods forproducing a process-related impurity and/or product-relatedsubstance-reduced protein preparation from a mixture comprising aprotein of interest (i.e., a product) and at least one process-relatedimpurity and/or product-related substance by subjecting the mixture toat least one cation exchange separation step. In certain embodiments,the CEX step will occur after the above-described Protein A affinitystep.

The use of a cationic exchange material versus a anionic exchangematerial, such as those anionic exchange materials discussed in detailabove, is based on the local charges of the protein of interest in agiven solution. Therefore, it is within the scope of this invention toemploy a cationic exchange step prior to the use of an anionic exchangestep, or an anionic exchange step prior to the use of a cationicexchange step. Furthermore, it is within the scope of this invention toemploy only a cationic exchange step, only an anionic exchange step, orany serial combination of the two (including serial combinations of oneor both ion exchange steps with the other chromatographic separationtechnologies described herein).

In performing the separation, the initial protein mixture can becontacted with the cation exchange material by using any of a variety oftechniques, e.g., using a batch purification technique or achromatographic technique, as described above in connection with ProteinA or AEX.

In certain embodiments, the aqueous salt solution used as both theloading and wash buffer has a pH that is lower than the isoelectricpoint (pI) of the protein of interest. In certain embodiments, the pH isabout 0 to 5 units lower than the pI of the protein. In certainembodiments, it is in the range of 1 to 2 units lower. In certainembodiments, it is in the range of 1 to 1.5 units lower.

In certain embodiments, the concentration of the anionic agent inaqueous salt solution is increased or decreased to achieve a pH ofbetween about 3.5 and 10.5, or between about 4 and 10, or between about4.5 and 9.5, or between about 5 and 9, or between about 5.5 and 8.5, orbetween about 6 and 8, or between about 6.5 and 7.5. In certainembodiments, the concentration of anionic agent is increased ordecreased in the aqueous salt solution to achieve a pH of 5, or 5.5, or6, or 6.5, or 6.8, or 7.5.

In certain embodiments, the conductivity and pH of the aqueous saltsolution is adjusted by increasing or decreasing the concentration of acationic agent. In certain embodiments, the cationic agent is maintainedat a concentration of between about range of 20 mM to 500 mM, or betweenabout 50 to 350 mM or between about 100 to 300 mM or between about 100to 200 mM.

In certain non-limiting embodiments, the cationic agent is selected fromthe group consisting of sodium, Tris, tromethalmine, ammonium, arginine,or combinations thereof. In certain non-limiting embodiments, theanionic agent is selected from the group consisting of acetate, citrate,chloride anion, sulphate, phosphate or combinations thereof.

A packed cation-exchange chromatography column or a cation-exchangemembrane device can be operated either in bind-elute mode, flow-throughmode, or a hybrid mode wherein the product exhibits binding to thechromatographic material, yet can be washed from the column using abuffer that is the same or substantially similar to the loading buffer.The details of these modes are outlined above.

Cationic substituents include carboxymethyl (CM), sulfoethyl (SE),sulfopropyl (SP), phosphate (P) and sulfonate (S). Additional cationicmaterials include, but are not limited to: Capto SP ImpRes, which is ahigh flow agarose bead; CM Hyper D grade F; which is a ceramic beadcoated and permeated with a functionalized hydrogel, 250-400 ionicgroups μeq/mL; Eshmuno S, which is a hydrophilic polyvinyl ether basematrix with 50-100 μeq/mL ionic capacity; Nuvia C Prime, which is ahydrophobic cation exchange media composed of a macroporous highlycrosslinked hydrophilic polymer matrix 55-75 uteq/mL; Nuvia S, which hasa UNOsphere base matrix with 90-150 μeq/mL ionic groups; Poros HS; whichis a rigid polymetic bead with a backbone consisting of cross-linkedpoly[styrene-divinylbenzene]; Poros XS; which is a rigid polymetic beadwith a backbone consisting of cross-linked poly[styrene-divinylbenzene];Toyo Pearl Giga Cap CM 650M, which is a polymeric base bead with 0.225meq/mL ionic capacity; Toyo Pearl Giga Cap S 650M which is a polymericbase bead; Toyo Pearl MX TRP, which is a polymeric base bead. Detailedinformation concerning the aforementioned materials is listed in Table2.

TABLE 2 Cationic Materials particle Catalog Resin Vendor type sizeNumber Capto SP ImpRes GE Strong ~40 μm 17-5468-10 CM Hyper D Pail Weak~50 μm 20050-027 Eshmuno S Millipore Strong ~85 μm 1.20078 Nuvia C PrimeBiorad Mix Mode ~70 μm 156-3401 Nuvia S Biorad Strong ~85 μm 156-0315Poros HS Applied Weak ~50 μm 13359-06 Biosystems Poros XS Applied Strong~50 μm 4404337 Biosystems Toyo Pearl Giga Tosoh Weak ~75 μm 21946 Cap CM650M Toyo Pearl Giga Tosoh Strong ~75 μm 21833 Cap S 650M Toyo PearlTosoh Mix Mode ~75 μm 22817 MX Trp 650M

In certain embodiments, the protein load of the mixture comprisingprotein of interest is adjusted to a total protein load to the column ofbetween about 5 and 150 g/L, or between about 10 and 100 g/L, betweenabout 20 and 80 g/L, or between about 30 and 50 g/L. In certainembodiments, the protein concentration of the load protein mixture isadjusted to a protein concentration of the material loaded to the columnof about 0.5 and 50 g/L, or between about 1 and 20 g/L.

In certain embodiments, additives such as poly ethylene glycol,detergents, amino acids, sugars, chaotropic agents can be added toenhance the performance of the separation, so as to achieve betterrecovery or product quality.

In certain embodiments, including, but not limited to those relating toAdalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR charge variants in the elution fractions while enriching for the samein the flow through and wash fractions, thereby producing proteinpreparations with reduced or free of AR variants. In certain embodimentsrelating to the purification of Adalimumab, the methods of the instantinvention can be used to selectively remove, significantly reduce, oressentially remove all of AR1 charge variants in the elution fractionswhile enriching for the same in the flow through and wash fractions,thereby producing protein preparations with reduced or free of AR1variants. In certain embodiments relating to the purification ofAdalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR2 charge variants in the elution fractions while enriching for thesame in the flow through and wash fractions, thereby producing proteinpreparations with reduced or free of AR2 variants.

In certain embodiments, including, but not limited to those relating toAdalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofthe Methyl Glycoxol (MGO) variants in the elution fractions whileenriching for the same in the flow through and wash fractions, therebyproducing protein preparations with reduced or free of MGO variants. Incertain embodiments, including, but not limited to those relating toAdalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofthe glycated variants (schiffs base and permanently glycated forms) inthe elution fractions while enriching for the same in the flow throughand wash fractions, thereby producing protein preparations with reducedor free of glycated variants.

In certain embodiments, the loading, pH, conductivity of the CEXchromatography step, as well as elution pH conductivity, can be modifiedto achieve a desired distribution of process-related impurities and/orproduct-relates substances. For example, but not by way of limitation,certain embodiments are directed to the modulation of the lysinedistribution of a purified sample of a protein of interest, e.g.,increasing Lys0 and decreasing Lys1 and Lys2. In certain embodiments,the methods of the present invention allow for the preparation ofsamples wherein the amount of Lys0 is decreased, while the amount ofLys1 and/or Lys2 is increased.

In certain embodiments, a CEX chromatographic separation can beperformed and combinations of fractions can be pooled to achieve acombination of desired process-related impurity and/or product-relatessubstance levels, in addition to, or in place of merely modulatingcharge variant concentration.

In certain embodiments, spectroscopy methods such as UV, NIR, FTIR,Fluorescence, Raman may be used to monitor levels of product-relatedcharge variants, aggregates, low molecular weight variants (e.g.,fragments of the protein of interest) in an on-line, at-line or in-linemode, which can then be used to control the level of charge variants,e.g., acidic species, in the pooled material collected from the CEXeffluent. In certain embodiments, specific signals arising from thechemical modification of the proteins such as glycation, MGOmodification, deamidation, glycosylation may be specifically measurableby spectroscopic methods through such in-line, on-line or at-linemethods, enabling realtime or near-real time control of product qualityof the resulting product. In certain embodiments, on-line, at-line orin-line monitoring methods can be used either on the effluent line ofthe chromatography step or in the collection vessel, to enableachievement of the desired product quality/recovery. In certainembodiments, the UV signal can be used as a surrogate to achieve anappropriate product quality/recovery, wherein the UV signal can beprocessed appropriately, including, but not limited to, such processingtechniques as integration, differentiation, moving average, such thatnormal process variability can be addressed and the target productquality can be achieved. In certain embodiments, such measurements canbe combined with in-line dilution methods such that ionconcentration/conductivity of the load/wash can be controlled byfeedback and hence facilitate product quality control.

In certain embodiments, a combination of CEX and AEX and MM methods canbe used to prepare product-related substance-modulated materials,including certain embodiments where one technology is used in acomplementary/supplementary manner with another technology. In certainembodiments, such a combination can be performed such that certainsub-species are removed predominantly by one technology, such that thecombination provides the desired final composition/product quality. Incertain embodiments, such combinations include the use of additionalintervening chromatography, filtration, pH adjustment, UF/DF steps so asto achieve the desired product quality, ion concentration, and/or viralreduction.

5.4.7. Mixed Mode Chromatography

Mixed mode (“MM”) chromatography, also referred to herein as “multimodalchromatography”, is a chromatographic strategy that utilizes a supportcomprising a ligand that is capable of providing at least two different,in certain embodiments co-operative, sites that interact with thesubstance to be bound. In certain embodiments, one of these sites givesan attractive type of charge-charge interaction between the ligand andthe substance of interest and the other site provides for electronacceptor-donor interaction and/or hydrophobic and/or hydrophilicinteractions. Electron donor-acceptor interactions include interactionssuch as hydrogen-bonding, π-π, cation-π, charge transfer, dipole-dipole,induced dipole etc.

In certain embodiments, the resin employed for a mixed mode separationis Capto Adhere. Capto Adhere is a strong anion exchanger withmultimodal functionality. Its base matrix is a highly cross-linkedagarose with a ligand (N-Benzyl-N-methyl ethanol amine) that exhibitsmany functionalities for interaction, such as ionic interaction,hydrogen bonding and hydrophobic interaction. In certain embodiments,the resin employed for a mixed mode separation is selected fromPPA-HyperCel and HEA-HyperCel. The base matrices of PPA-HyperCel andHEA-HyperCel are high porosity cross-linked cellulose. Their ligands arePhenylpropylamine and Hexylamine, respectively. Phenylpropylamine andHexylamine offer different selectivity and hydrophobicity options forprotein separations. Additional mixed mode chromatographic supportsinclude, but are not limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M,and Eshmuno® HCX.

In certain embodiments, the mixed mode chromatography resin is comprisedof ligands coupled to an organic or inorganic support, sometimes denoteda base matrix, directly or via a spacer. The support may be in the formof particles, such as essentially spherical particles, a monolith,filter, membrane, surface, capillaries, etc. In certain embodiments, thesupport is prepared from a native polymer, such as cross-linkedcarbohydrate material, such as agarose, agar, cellulose, dextran,chitosan, konjac, carrageenan, gellan, alginate etc. To obtain highadsorption capacities, the support can be porous, and ligands are thencoupled to the external surfaces as well as to the pore surfaces. Suchnative polymer supports can be prepared according to standard methods,such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta79(2), 393-398 (1964). Alternatively, the support can be prepared from asynthetic polymer, such as cross-linked synthetic polymers, e.g. styreneor styrene derivatives, divinylbenzene, acrylamides, acrylate esters,methacrylate esters, vinyl esters, vinyl amides etc. Such syntheticpolymers can be produced according to standard methods, see e.g.“Styrene based polymer supports developed by suspension polymerization”(R Arshady: Chimica e L′Industria 70(9), 70-75 (1988)). Porous native orsynthetic polymer supports are also available from commercial sources,such as Amersham Biosciences, Uppsala, Sweden.

In certain embodiments, the protein load of the mixture comprisingprotein of interest is adjusted to a total protein load to the column ofbetween about 50 and 750 g/L, or between about 75 and 500 g/L, orbetween about 100 and 300 g/L. In certain embodiments, the proteinconcentration of the load protein mixture is adjusted to a proteinconcentration of the material loaded to the column of about 1 and 50g/L, or between about 9 and 25 g/L.

In certain embodiments, additives such as poly ethylene glycol,detergents, amino acids, sugars, chaotropic agents can be added toenhance the performance of the separation, so as to achieve betterrecovery or product quality.

In certain embodiments, including, but not limited to those relating toAdalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR charge variants in the flow through and wash fractions whileenriching for the same in the flow elution fraction, thereby producingprotein preparations with reduced or free of AR variants. In certainembodiments relating to Adalimumab, the methods of the instant inventioncan be used to selectively remove, significantly reduce, or essentiallyremove all of AR1 charge variants in the flow through and wash fractionswhile enriching for the same in the flow elution fraction, therebyproducing protein preparations with reduced or free of AR1 variants. Incertain embodiments relating to Adalimumab, the methods of the instantinvention can be used to selectively remove, significantly reduce, oressentially remove all of AR2 charge variants in the flow-through andwash fractions while enriching for the same in the flow elutionfraction, thereby producing protein preparations with reduced or free ofAR2 variants.

In certain embodiments, including, but not limited to those relating toAdalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofthe methylglyoxal (MGO) variants in the flow through and wash fractionswhile enriching for the same in the elution fraction, thereby producingprotein preparations with reduced or free of MGO variants. In certainembodiments, including, but not limited to those relating to Adalimumab,the methods of the instant invention can be used to selectively remove,significantly reduce, or essentially remove all of the glycated variants(schiff's base and permanently glycated forms) in the flow through andwash fractions while enriching for the same in the elution fraction,thereby producing protein preparations with reduced or free of glycatedvariants.

In certain embodiments, the loading, pH, conductivity of the MMchromatography step, wash pH and conductivity, as well as elution pHconductivity, can be modified to achieve a desired distribution ofprocess-related impurities and/or product-relates substances. Forexample, but not by way of limitation, certain embodiments are directedto the modulation of the lysine distribution of a purified sample of aprotein of interest, e.g., increasing Lys0 and decreasing Lys1 and Lys2.In certain embodiments, the methods of the present invention allow forthe preparation of samples wherein the amount of Lys0 is decreased,while the amount of Lys1 and/or Lys2 is increased.

In certain embodiments, a MM chromatographic separation can be performedand combinations of fractions can be pooled to achieve a combination ofdesired process-related impurity and/or product-relates substancelevels, in addition to, or in place of merely modulating charge variantconcentration.

In certain embodiments, spectroscopy methods such as UV, NIR, FTIR,Fluorescence, Raman may be used to monitor levels of product-relatedcharge variants, aggregates, low molecular weight variants (e.g.,fragments of the protein of interest) in an on-line, at-line or in-linemode, which can then be used to control the level of charge variants,e.g., acidic species, in the pooled material collected from the MMeffluent. In certain embodiments, specific signals arising from thechemical modification of the proteins such as glycation, MGOmodification, deamidation, glycosylation may be specifically measurableby spectroscopic methods through such in-line, on-line or at-linemethods, enabling realtime or near-real time control of product qualityof the resulting product. In certain embodiments, on-line, at-line orin-line monitoring methods can be used either on the effluent line ofthe chromatography step or in the collection vessel, to enableachievement of the desired product quality/recovery. In certainembodiments, the UV signal can be used as a surrogate to achieve anappropriate product quality/recovery, wherein the UV signal can beprocessed appropriately, including, but not limited to, such processingtechniques as integration, differentiation, moving average, such thatnormal process variability can be addressed and the target productquality can be achieved. In certain embodiments, such measurements canbe combined with in-line dilution methods such that ionconcentration/conductivity of the load/wash can be controlled byfeedback and hence facilitate product quality control

In certain embodiments, a combination of mixed mode and AEX and CEXmethods can be used to prepare product-related charge variant-reducedmaterials, including certain embodiments where one technology is used ina complementary/supplementary manner with another technology. In certainembodiments, such a combination can be performed such that certainsub-species are removed predominantly by one technology, such that thecombination provides the desired final composition/product quality. Incertain embodiments, such combinations include the use of additionalintervening chromatography, filtration, pH adjustment, UF/DF steps so asto achieve the desired product quality, ion concentration, and/or viralreduction.

5.4.8. Hydrophobic Interaction Chromatography

The present invention also features methods for producing aprocess-related impurity and/or product-related substance-reducedprotein preparation from a mixture comprising a protein of interest,e.g., an antibody, and at least one process-related impurity and/orproduct-related substance further comprising a hydrophobic interactionchromatography (HIC) step in addition to the displacement chromatographystep.

In performing the separation, the sample mixture is contacted with theHIC material, e.g., using a batch purification technique or using acolumn or membrane chromatography. Prior to HIC purification it may bedesirable to adjust the concentration of the salt buffer to achievedesired protein binding to the resin or the membrane.

Whereas ion exchange chromatography relies on the local charge of theprotein of interest for selective separation, hydrophobic interactionchromatography employs the hydrophobic properties of the proteins toachieve selective separation. Hydrophobic groups on the protein interactwith hydrophobic groups of the resin or the membrane. The morehydrophobic a protein is the stronger it will interact with the columnor the membrane. Thus the HIC step removes process-related impurities(e.g., HCPs) as well as product-related substances (e.g., aggregates andfragments).

Like ion exchange chromatography, a HIC column or membrane device canalso be operated in product a bind-elute mode, a flow-through, or ahybrid mode wherein the product exhibits binding to the chromatographicmaterial, yet can be washed from the column using a buffer that is thesame or substantially similar to the loading buffer. The details ofthese modes are outlined above in connection with AEX purification.

As hydrophobic interactions are strongest at high ionic strength, thisform of separation is conveniently performed following salt elutionstep, such as those that are typically used in connection with ionexchange chromatography. Alternatively, salts can be added into a lowsalt level feed stream before this step. Adsorption of the antibody to aHIC column is favored by high salt concentrations, but the actualconcentrations can vary over a wide range depending on the nature of theprotein of interest, salt type and the particular HIC ligand chosen.Various ions can be arranged in a so-called soluphobic series dependingon whether they promote hydrophobic interactions (salting-out effects)or disrupt the structure of water (chaotropic effect) and lead to theweakening of the hydrophobic interaction. Cations are ranked in terms ofincreasing salting out effect as Ba²⁺; Ca²⁺; Mg²⁺; Li⁺; Cs⁺; Na⁺; K⁺;Rb⁺; NH₄ ⁺, while anions may be ranked in terms of increasing chaotropiceffect as PO₄ ³⁻; SO₄ ²⁻; CH₃CO₃ ⁻; Cl⁻; Br⁻; NO₃ ⁻; ClO₄ ⁻; I⁻; SCN⁻.

In general, Na⁺, K⁺ or NH₄ ⁺ sulfates effectively promote ligand-proteininteraction in HIC. Salts may be formulated that influence the strengthof the interaction as given by the following relationship:(NH₄)₂SO₄>Na₂SO₄>NaCl>NH₄Cl>NaBr>NaSCN. In general, salt concentrationsof between about 0.75 M and about 2 M ammonium sulfate or between about1 and 4 M NaCl are useful.

HIC media normally comprise a base matrix (e.g., cross-linked agarose orsynthetic copolymer material) to which hydrophobic ligands (e.g., alkylor aryl groups) are coupled. A suitable HIC media comprises an agaroseresin or a membrane functionalized with phenyl groups (e.g., a PhenylSepharose™ from GE Healthcare or a Phenyl Membrane from Sartorius). ManyHIC resins are available commercially. Examples include, but are notlimited to, Capto Phenyl, Phenyl Sepharose™ 6 Fast Flow with low or highsubstitution, Phenyl Sepharose™ High Performance, Octyl Sepharose™ HighPerformance (GE Healthcare); Fractogel™ EMD Propyl or Fractogel™ EMDPhenyl (E. Merck, Germany); Macro-Prep™ Methyl or Macro-Prep™ t-Butylcolumns (Bio-Rad, California); WP HI-Propyl (C3)™ (J. T. Baker, NewJersey); and Toyopearl™ ether, phenyl or butyl (TosoHaas, PA).

5.4.9. Viral Filt Ration

Viral filtration is a dedicated viral reduction step in the entirepurification process. This step is usually performed postchromatographic polishing steps. Viral reduction can be achieved via theuse of suitable filters including, but not limited to, Planova 20N™, 50N or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMDMillipore, ViroSart CPV from Sartorius, or Ultipor DV20 or DV50™ filterfrom Pall Corporation. It will be apparent to one of ordinary skill inthe art to select a suitable filter to obtain desired filtrationperformance.

5.4.10. Ultrafiltration/Diafiltration

Certain embodiments of the present invention employ ultrafiltration anddiafiltration steps to further concentrate and formulate the protein ofinterest, e.g., an antibody product. Ultrafiltration is described indetail in: Microfiltration and Ultrafiltration: Principles andApplications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York,N.Y., 1996); and in: Ultrafiltration Handbook, Munir Cheryan (TechnomicPublishing, 1986; ISBN No. 87762-456-9). One filtration process isTangential Flow Filtration as described in the Millipore catalogueentitled “Pharmaceutical Process Filtration Catalogue” pp. 177-202(Bedford, Mass., 1995/96). Ultrafiltration is generally considered tomean filtration using filters with a pore size of smaller than 0.1 μm.By employing filters having such small pore size, the volume of thesample can be reduced through permeation of the sample buffer throughthe filter membrane pores while proteins, such as antibodies, areretained above the membrane surface.

Diafiltration is a method of using membrane filters to remove andexchange salts, sugars, and non-aqueous solvents, to separate free frombound species, to remove low molecular-weight species, and/or to causethe rapid change of ionic and/or pH environments. Microsolutes areremoved most efficiently by adding solvent to the solution beingdiafiltered at a rate approximately equal to the permeate flow rate.This washes away microspecies from the solution at a constant volume,effectively purifying the retained protein of interest. In certainembodiments of the present invention, a diafiltration step is employedto exchange the various buffers used in connection with the instantinvention, optionally prior to further chromatography or otherpurification steps, as well as to remove impurities from the proteinpreparations.

One of ordinary skill in the art can select appropriate membrane filterdevice for the UF/DF operation. Examples of membrane cassettes suitablefor the present invention include, but not limited to, Pellicon 2 orPellicon 3 cassetts with 10 kD, 30 kD or 50 kD membranes from EMDMillipore, Kvick 10 kD, 30 kD or 50 kD membrane cassettes from GEHealthcare, and Centramate or Centrasette 10 kD, 30 kD or 50 kDcassettes from Pall Corporation.

5.4.11. Exemplary Purification Strategies

In certain embodiments, primary recovery can proceed by sequentiallyemploying pH reduction, centrifugation, and filtration steps to removecells and cell debris (including HCPs) from the production bioreactorharvest. In certain embodiments, the present invention is directed tosubjecting a sample mixture from said primary recovery to one or moreAEX, CEX, and/or MM purification steps. Certain embodiments of thepresent invention will include further purification steps. Examples ofadditional purification procedures which can be performed prior to,during, or following the ion exchange chromatography method includeethanol precipitation, isoelectric focusing, reverse phase HPLC,chromatography on silica, chromatography on heparin Sepharose™, furtheranion exchange chromatography and/or further cation exchangechromatography, chromatofocusing, SDS-PAGE, ammonium sulfateprecipitation, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography (e.g., using protein G, anantibody, a specific substrate, ligand or antigen as the capturereagent).

Specific examples of such combinations of strategies is presented below,with specific data relating to particular combinations useful in thecontext of the instant invention included in Tables 44-51 and 72-74.

In certain embodiments the unbound flow through and wash fractions canbe further fractionated and a combination of fractions providing atarget product purity can be pooled.

In certain embodiments the protein concentration can be adjusted toachieve a differential partitioning behavior between the antibodyproduct and the product-related substances such that the purity and/oryield can be further improved. In certain embodiments the loading can beperformed at different protein concentrations during the loadingoperation to improve the product quality/yield of any particularpurification step.

In certain embodiments the column temperature, can be independentlyvaried to improve the separation efficiency and/or yield of anyparticular purification step.

In certain embodiments, the loading and washing buffer matrices can bedifferent or composed of mixtures of chemicals, while achieving similar“resin interaction” behavior such that the above novel separation can beeffected. For example, but not by way of limitation, the loading andwashing buffers can be different, in terms of ionic strength or pH,while remaining substantially similar in function in terms of thewashout of the product achieved during the wash step. In certainembodiments, additives such as amino acids, sugars, PEG, etc can beadded to the load or wash steps to modulate the partitioning behavior toachieve the separation efficiency and/or yield.

In certain embodiments, the loading & washing steps can be controlled byin-line, at-line or off-line measurement of the product relatedimpurity/substance levels, either in the column effluent, or thecollected pool or both, so as to achieve the target product qualityand/or yield. In certain embodiments, the loading concentration can bedynamically controlled by in-line or batch or continuous dilutions withbuffers or other solutions to achieve the partitioning necessary toimprove the separation efficiency and/or yield.

5.5. Methods of Assaying Sample Purity

5.5.1. Assaying Host Cell Protein

The present invention also provides methods for determining the residuallevels of host cell protein (HCP) concentration in the isolated/purifiedantibody composition. As described above, HCPs are desirably excludedfrom the final target substance product. Exemplary HCPs include proteinsoriginating from the source of the antibody production. Failure toidentify and sufficiently remove HCPs from the target antibody may leadto reduced efficacy and/or adverse subject reactions.

As used herein, the term “HCP ELISA” refers to an ELISA where the secondantibody used in the assay is specific to the HCPs produced from cells,e.g., CHO cells, used to generate the antibody of interest. The secondantibody may be produced according to conventional methods known tothose of skill in the art. For example, the second antibody may beproduced using HCPs obtained by sham production and purification runs,i.e., the same cell line used to produce the antibody of interest isused, but the cell line is not transfected with antibody DNA. In anexemplary embodiment, the second antibody is produced using HCPs similarto those expressed in the cell expression system of choice, i.e., thecell expression system used to produce the target antibody.

Generally, HCP ELISA comprises sandwiching a liquid sample comprisingHCPs between two layers of antibodies, i.e., a first antibody and asecond antibody. The sample is incubated during which time the HCPs inthe sample are captured by the first antibody, for example, but notlimited to goat anti-CHO, affinity purified (Cygnus). A labeled secondantibody, or blend of antibodies, specific to the HCPs produced from thecells used to generate the antibody, e.g., anti-CHO HCP Biotinylated, isadded, and binds to the HCPs within the sample. In certain embodimentsthe first and second antibodies are polyclonal antibodies. In certainaspects the first and second antibodies are blends of polyclonalantibodies raised against HCPs. The amount of HCP contained in thesample is determined using the appropriate test based on the label ofthe second antibody.

HCP ELISA may be used for determining the level of HCPs in an antibodycomposition, such as an eluate or flow-through obtained using theprocess described above. The present invention also provides acomposition comprising an antibody, wherein the composition has nodetectable level of HCPs as determined by an HCP Enzyme LinkedImmunosorbent Assay (“ELISA”).

5.5.2. Assaying Charge Variants and Aggregates

In certain embodiments, the levels of acidic species and other chargevariants in the chromatographic samples produced using the techniquesdescribed herein are analyzed. In certain embodiments a CEX-HPLC methodis employed. For example, but not by way of limitation, cation exchangechromatography can be performed on a Dionex ProPac WCX-10, Analyticalcolumn 4 mm×250 mm (Dionex, Calif.). An Agilent 1200 HPLC system canthen be used as the HPLC. In certain embodiments, mobile phases such as10 mM Sodium Phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM SodiumPhosphate dibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B) can beused. In certain embodiments, a binary gradient (94% A, 6% B: 0-20 min;84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% A, 6% B: 28-34min) can be used with detection at 280 nm. In certain embodiments,quantitation is based on the relative area percent of detected peaks. Incertain embodiments, the peaks that elute at relative residence timeless than a certain time are together represented as the acidic peaks.

In certain embodiments, the levels of aggregates, monomer, and fragmentsin the chromatographic samples produced using the techniques describedherein are analyzed. In certain embodiments, the aggregates, monomer,and fragments are measured using a size exclusion chromatographic (SEC)method for each molecule. For example, but not by way of limitation, aTSK-gel G3000SWxL, 5 μm, 125 Å, 7.8×300 mm column (Tosoh Bioscience) canbe used in connection with certain embodiments, while a TSK-gel SuperSW3000, 4 μm, 250 Å, 4.6×300 mm column (Tosoh Bioscience) can be used inalternative embodiments. In certain embodiments, the aforementionedcolumns are used along with an Agilent or a Shimazhu HPLC system. Incertain embodiments, sample injections are made under isocratic elutionconditions using a mobile phase consisting of, for example, 100 mMsodium sulfate and 100 mM sodium phosphate at pH 6.8, and detected withUV absorbance at 214 nm. In certain embodiments, the mobile phase willconsist of 1×PBS at pH 7.4, and elution profile detected with UVabsorbance at 280 nm. In certain embodiments, quantification is based onthe relative area of detected peaks.

5.6. Further Modifications

The purified proteins, e.g., antibodies and antibody-binding portionsthereof, of the present invention can be modified. In some embodiments,the antibodies are chemically modified to provide a desired effect. Forexample, but not by way of limitation, pegylation of antibodies orantibody fragments of the invention may be carried out by any of thepegylation reactions known in the art, as described, e.g., in thefollowing references: Focus on Growth Factors 3:4-10 (1992); EP 0 154316; and EP 0 401 384, each of which is incorporated by reference hereinin its entirety. In one aspect, the pegylation is carried out via anacylation reaction or an alkylation reaction with a reactivepolyethylene glycol molecule (or an analogous reactive water-solublepolymer). A suitable water-soluble polymer for pegylation of theantibodies and antibody fragments of the invention is polyethyleneglycol (PEG). As used herein, “polyethylene glycol” is meant toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono (Cl—ClO) alkoxy- or aryloxy-polyethyleneglycol.

Methods for preparing pegylated antibodies and antibody fragments of theinvention will generally comprise the steps of (a) reacting the antibodyor antibody fragment with polyethylene glycol, such as a reactive esteror aldehyde derivative of PEG, under suitable conditions whereby theantibody or antibody fragment becomes attached to one or more PEGgroups, and (b) obtaining the reaction products. It will be apparent toone of ordinary skill in the art to select the optimal reactionconditions or the acylation reactions based on known parameters and thedesired result.

Generally the pegylated antibodies and antibody fragments have increasedhalf-life, as compared to the nonpegylated antibodies and antibodyfragments. The pegylated antibodies and antibody fragments may beemployed alone, together, or in combination with other pharmaceuticalcompositions.

An antibody of the invention can be derivatized or linked to anotherfunctional molecule (e.g., another peptide or protein). For example, anantibody of the invention can be functionally linked (by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other molecular entities, such as another antibody (e.g., abispecific antibody or a diabody), a detectable agent, a cytotoxicagent, a pharmaceutical agent, and/or a protein or peptide that canmediate associate of the antibody with another molecule (such as astreptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an antibody of the invention may bederivatized include fluorescent compounds. Exemplary fluorescentdetectable agents include fluorescein, fluorescein isothiocyanate,rhodanine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrinand the like. An antibody may also be derivatized with detectableenzymes, such as alkaline phosphatase, horseradish peroxidase, glucoseoxidase and the like. When an antibody is derivatized with a detectableenzyme, it is detected by adding additional reagents that the enzymeuses to produce a detectable reaction product. For example, when thedetectable agent horseradish peroxidase is present, the addition ofhydrogen peroxide and diaminobenzidine leads to a colored reactionproduct, which is detectable. An antibody may also be derivatized withbiotin, and detected through indirect measurement of avidin orstreptavidin binding.

5.7. Pharmaceutical Compositions

The proteins of interest, e.g., antibodies and antibody-binding portionsthereof, of the invention can be incorporated into pharmaceuticalcompositions suitable for administration to a subject. In certainembodiments, the pharmaceutical composition comprises an antibody of theinvention and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it is desirable to include isotonicagents, e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride in the composition. Pharmaceutically acceptable carriers mayfurther comprise minor amounts of auxiliary substances such as wettingor emulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the antibody.

The antibodies and antibody-binding portions thereof, of the inventioncan be incorporated into a pharmaceutical composition suitable forparenteral administration. The antibody or antibody-portions can beprepared as an injectable solution containing, e.g., 0.1-250 mg/mLantibody. The injectable solution can be composed of either a liquid orlyophilized dosage form in a flint or amber vial, ampule or pre-filledsyringe. The buffer can be L-histidine approximately 1-50 mM, (optimally5-10 mM), at pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffersinclude but are not limited to sodium succinate, sodium citrate, sodiumphosphate or potassium phosphate. Sodium chloride can be used to modifythe toxicity of the solution at a concentration of 0-300 mM (optimally150 mM for a liquid dosage form). Cryoprotectants can be included for alyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%).Other suitable cryoprotectants include trehalose and lactose. Bulkingagents can be included for a lyophilized dosage form, principally 1-10%mannitol (optimally 24%). Stabilizers can be used in both liquid andlyophilized dosage forms, principally 1-50 mM L-methionine (optimally5-10 mM). Other suitable bulking agents include glycine, arginine, canbe included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).Additional surfactants include but are not limited to polysorbate 20 andBRIJ surfactants.

In one aspect, the pharmaceutical composition includes the antibody at adosage of about 0.01 mg/kg-10 mg/kg. In another aspect, the dosages ofthe antibody include approximately 1 mg/kg administered every otherweek, or approximately 0.3 mg/kg administered weekly. A skilledpractitioner can ascertain the proper dosage and regime foradministering to a subject.

The compositions of this invention may be in a variety of forms. Theseinclude, e.g., liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Theform depends on, e.g., the intended mode of administration andtherapeutic application. Typical compositions are in the form ofinjectable or infusible solutions, such as compositions similar to thoseused for passive immunization of humans with other antibodies. One modeof administration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In one aspect, the antibody isadministered by intravenous infusion or injection. In another aspect,the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile, lyophilized powders for thepreparation of sterile injectable solutions, the methods of preparationare vacuum drying and spray-drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, e.g., by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, e.g., monostearate salts and gelatin.

The antibodies and antibody-binding portions thereof, of the presentinvention can be administered by a variety of methods known in the art,one route/mode of administration is subcutaneous injection, intravenousinjection or infusion. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, theentire teaching of which is incorporated herein by reference.

In certain aspects, an antibody or antibody-binding portion thereof, ofthe invention may be orally administered, e.g., with an inert diluent oran assimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain aspects, an antibody or antibody-bindingportion thereof, of the invention is co-formulated with and/orco-administered with one or more additional therapeutic agents that areuseful for treating disorders. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies. It will be appreciated by the skilledpractitioner that when the antibodies of the invention are used as partof a combination therapy, a lower dosage of antibody may be desirablethan when the antibody alone is administered to a subject (e.g., asynergistic therapeutic effect may be achieved through the use ofcombination therapy which, in turn, permits use of a lower dose of theantibody to achieve the desired therapeutic effect).

It should be understood that the antibodies of the invention can be usedalone or in combination with an additional agent, e.g., a therapeuticagent, said additional agent being selected by the skilled artisan forits intended purpose. For example, the additional agent can be atherapeutic agent art-recognized as being useful to treat the disease orcondition being treated by the antibody of the present invention. Theadditional agent also can be an agent which imparts a beneficialattribute to the therapeutic composition, e.g., an agent which affectsthe viscosity of the composition.

6. EXAMPLES 6.1. Anion Exchange Chromatography Examples 6.1.1. Materials& Methods

6.1.1.1. Chromatography Method

Except where noted, the materials and methods described in connectionwith the instant example were also employed in the examples of Sections6.2., 6.3., and 6.4., below.

Pre-packed resin columns were used in the following experiments, exceptwhere specified. The column was equilibrated in a buffer system withappropriate pH and conductivity. The column load was prepared fromProtein A affinity chromatography eluates or concentrated CEXchromatography elutes by buffer exchange (if the eluates were withdifferent buffer components from the mixed mode target buffer system) oraddition of the stock solutions and/or water to obtain the target pH andconductivity as specified (if the eluates were with the same buffercomponents as the mixed mode target buffer system). The prepared loadmaterial was filtered and loaded on the column according to the targetload amount (g protein/L resin) as specified followed by washing withthe equilibration buffer or buffer similar to equilibration buffer withvolumes as specified. The column Flow Through/Wash were collected asfractions or as a pool. Mixed mode column was regenerated with 0.1Macetic acid, 0.15M NaCl pH3, or 0.1M Acetic acid solution, pH 3, or asspecified. 1M NaOH solution was used for column cleaning.

6.1.1.2. Buffer Preparation Method

Buffers were prepared targeting specific ion concentration for the anionby fixing the anion concentration (acid) to the target value, andadjusting the solution with the cationic component (base) to achieve theappropriate pH. For example to prepare a 10 mM Acetate-Tris buffersolution, pH 8.7, glacial acetic acid was dissolved in water to a targetconcentration of 10 mM and adjusted with concentrated Tris-base to pH8.7.

6.1.1.3. AR Reduction and Recovery Calculations

In general, the Flow Through/Wash fractions were collected and analyzedwith WCX-10 method for AR levels. By actual or calculated pooling of thefractions the recovery and the corresponding AR levels were calculated.

6.1.1.4. WCX-10 for Adalimumab

The acidic species and other charge variants present in the Adalimumabprocess samples were quantified according to the following methods.Cation exchange chromatography was performed on a Dionex ProPac WCX-10,Analytical column 4 mm×250 mm (Dionex, Calif.). An Agilent 1200 HPLCsystem was used as the HPLC. The mobile phases used were 10 mM SodiumPhosphate dibasic pH 7.5 (Mobile phase A) and 10 mM Sodium Phosphatedibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B). A binarygradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B:22-28 min; 94% A, 6% B: 28-34 min) was used with detection at 280 nm.

Quantitation was based on the relative area percent of detected peaks.The peaks that elute at relative residence time less than a certain timeare together represented as the acidic peaks.

6.1.1.5. WCX-10 for mAb-B

The acidic species and other charge variants present in the mAb-Bprocess samples were quantified according to the following methods.Cation exchange chromatography was performed on a Dionex ProPac WCX-10,Analytical column 4 mm×250 mm (Dionex, Calif.). An Agilent 1200 HPLCsystem was used as the HPLC. The mobile phases used were 20 mM4-Morpholineethanesulfonic acid (MES), pH 6.5 (Mobile phase A) and 20 mM4-Morpholineethanesulfonic acid (MES), 500 mM Sodium Chloride pH 6.5(Mobile phase B). A binary gradient (87% A, 13% B: 0-5 min; 87% A, 13%B: 5-35 min; 75% A, 25% B: 35-40 min; 0% A, 100% B: 40-43 min; 87% A,13% B: 43-46 min; 87% A, 13% B: 46-55 min) was used with detection at280 nm, bw 8 nm; ref 360 nm, bw 100 nm.

Quantitation was based on the relative area percent of detected peaks.All peaks eluting prior to the Main Isoform peak were summed as theacidic region, and all peaks eluting after the LYS-2 peaks will besummed as the basic region.

6.1.1.6. WCX-10 for mAb-C

The mAb-C method was employed towards the quantification of the acidicspecies and other charge variants present mAb-C process samples. Cationexchange chromatography was performed on a Dionex ProPac WCX-10,Analytical column 4 mm×250 mm (Dionex, Calif.). An Agilent 1200 HPLCsystem was used as the HPLC. The mobile phases used were 20 mM4-Morpholineethanesulfonic acid (MES), pH 6.0 (Mobile phase A) and 20 mM4-Morpholineethanesulfonic acid (MES), 250 mM Sodium Chloride pH 6.0(Mobile phase B). A binary gradient (97% A, 3% B: 0-1 min; 79% A, 21% B:1-46 min; 0% A, 100% B: 46-47 min; 0% A, 100% B: 47-52 min; 97% A, 3% B:52-53 min; 97% A, 3% B: 53-60 min) was used with detection at 280 nm, bw8 nm; ref 360 nm, bw 100 nm.

Quantitation was based on the relative area percent of detected peaks.All peaks eluting prior to the Main Isoform peak will be summed as theacidic region, and all peaks eluting after the Main Isoform peak will besummed as the basic region.

6.1.1.7. Size Exclusion Chromatography

The molecular weight distribution of collected samples were quantifiedaccording to the following methods. Size exclusion chromatography (SEC)was performed using a TSK-gel G3000SWxL, 5 μm, 125 Å, 7.8×300 mm column(Tosoh Bioscience) on an HP Agilent HPLC system. Injections were madeunder isocratic elution conditions using a mobile phase of 200 mM sodiumsulfate, 100 mM sodium phosphate, pH 6.8, and detected with absorbanceat 214 nm. Quantification is based on the relative area of detectedpeaks.

6.1.1.8. Host Cell Protein (HCP) ELISA

HCP assay is based on process specific antigen based ELISA. Sampledilutions were applied to achieve readings within the calibration range.The limit of quantitation of the assay is 0.625 ng/mL.

6.1.1.9. UV spectroscopy A₂₈₀

UV A280 was used to determine protein concentrations for the samplespost protein A elution. The assay was performed on an Agilent UVSpectrophotometer following the method. The protein concentration wasdetermined using Beer-Lambert's Law, A=εlc, where A is Absorbance, E isthe extinction coefficient, l is the path length, and c is theconcentration. The absorbance was taken at 280 nm, the path length was 1cm, and the extinction coefficients were 1.39 for Adalimumab, 1.38 formAb B, and 1.43 for mAb C.

6.1.2 Example AEX 1 Determining Operating Conditions Appropriate for aMab: Media: Buffer Combination

The demonstration of the current invention for a specific antibody &resin is provided in this example, and consists of

-   -   1. Choosing an anion concentration that allows product and        impurities to bind at a given pH above the pI of the product.    -   2. Performing a pH gradient elution covering a range above, at,        and below the pI of the product.    -   3. Determining pH range in which the protein elutes from the        anion exchange media

In this example, adalimumab and Poros 50PI were chosen. The experimentwas performed at acetate (anion) concentration of 5 mM. The column wasequilibrated with 5 mM acetate/iTris at a pH of 9.0. Adalimumab wasprepared at 5 mM acetate/Tris pH 9.0 and loaded to the column at 20g-protein/L of resin. The column was washed with 10 CVs of theequilibration buffer. A pH gradient from 9.0 to 7.0 at an anionconcentration of 5 mM acetate/Tris was then performed. The processchromatograms are shown in FIG. 1.

The demonstration of the current invention for a specific antibody &resin is provided in this example, and consists of

-   -   1. For a given pH, choosing a starting anion concentration that        allows product and impurities to bind to the AEX adsorbent.    -   2. Loading a small amount of protein to the column and then        performing a linear gradient elution by increasing the anion        concentration keeping pH constant.    -   3. Determining anion concentration range in which the protein        elutes from the anion exchange media.

In this example, adalimumab and Poros 50HQ were chosen. The experimentwas performed at a pH 8.7. The column was equilibrated with 10 mMacetate/Tris at pH 8.7. Adalimumab was prepared at 10 mM acetate/Tris pH8.7 and loaded to the column at 20 g-protein/L of resin. The column waswashed with 10 CVs of the equilibration buffer. A linear gradient from10-100 mM Acetate/Tris at pH 8.7 was performed. The processchromatograms are shown in FIG. 2.

This general approach is used to determine the appropriate operatingcondition, example shown in Table 3, for any resin/mAb combination, toimplement the invention.

TABLE 3 Example Experimental Design Scope determined from pH and aniongradient elution Poros 50HQ - 300 g/L Loading - 30 g/L Fractionation pHRange 8.2-9.0 Anion Concentration (acetate) 10-20 mM

In practicing the current invention, the acidic species reductiondesired can be achieved by appropriate pooling of the load and washfractions. By collecting and subsequently determining the productquality of each fraction throughout the load and wash, the accumulativeAR reduction and accumulative yield can be calculated using the weightedaverages up to a given fraction. Additionally, the instantaneous yieldcan be estimated by comparing the protein recovered against the totalprotein loaded to the column at a given fraction. Sample calculationsare shown below:

Sample  Calculation  A:  Accumulative  Yield  up  to  a  given  fraction${{Accumulative}\mspace{14mu} {Yield}} = \frac{{Accumulated}\mspace{14mu} {Protein}\mspace{14mu} {Mass}\mspace{14mu} {Recovered}\mspace{14mu} {up}\mspace{14mu} {to}\mspace{14mu} {Fraction}}{{Total}\mspace{14mu} {Mass}\mspace{14mu} {Protein}\mspace{14mu} {Load}}$Sample  Calculation  B:  Accumulative  AR  Reduction  up  to  a  given  fraction${{Accumulative}\mspace{14mu} {AR}\mspace{14mu} {Reduction}} = {{{Load}\mspace{14mu} {AR}\mspace{14mu} \%} - \frac{{Accumulated}\mspace{14mu} {Acidic}\mspace{14mu} {Species}\mspace{14mu} {Mass}\mspace{14mu} {Recovered}\mspace{14mu} {up}\mspace{14mu} {to}\mspace{14mu} {Fraction}}{{Accumulated}\mspace{14mu} {Total}\mspace{14mu} {Protein}\mspace{14mu} {Mass}\mspace{14mu} {Recovered}\mspace{14mu} {up}\mspace{14mu} {to}\mspace{14mu} {Fraction}}}$Sample  Calculation  C:  Instantaneous  Yield  up  to  a  given  fraction${{Instantaneous}\mspace{14mu} {Yield}} = \frac{{Accumulated}\mspace{14mu} {Protein}\mspace{14mu} {Mass}\mspace{14mu} {Recovered}\mspace{14mu} {up}\mspace{14mu} {to}\mspace{14mu} {Fraction}}{{Total}\mspace{14mu} {Protein}\mspace{14mu} {Mass}\mspace{14mu} {Loaded}\mspace{14mu} {to}\mspace{14mu} {Column}\mspace{14mu} {at}\mspace{14mu} {Fraction}}$

The demonstration of the current invention for a specific antibody &resin is provided in this example, and consists of

-   -   1. For a given pH and anion concentration and anion exchange        media.    -   2. Loading the anion exchange media in excess of the dynamic        binding capacity for the product for the given condition.    -   3. Washing the column with a buffer containing a similar pH and        anion concentration used for the equilibration and loading        steps.    -   4. Collecting fractions throughout the loading and wash steps        and subsequently determining the product quality profile (e.g.        AR, aggregate, etc.)

In this example, adalimumab and Poros 50PI were chosen. The experimentwas performed at 5 mM acetate/arginine pH 8.8. The column wasequilibrated with 5 mM acetate/arginine at pH 8.8. Adalimumab wasprepared at 5 mM acetate/arginine pH 8.8 and loaded to the column at 300g-protein/L-resin. The column was washed with 20 CVs of theequilibration buffer. Fractions were collected in volumes representing30 g-protein/L-resin, shown in FIG. 3. Each fraction was then analyzedfor product quality and the accumulative yield and AR reductioncalculated, shown in Table 4. From this example, it is clear to oneskilled in the art to determine a run condition which delivers atargeted product quality and/or step yield.

This general approach is used to evaluate the performance for a givenoperating condition for any resin/mAb/buffer combination.

TABLE 4 Accumulative Yield and AR Reduction from FIG. 3 AccumulativeFraction Load Yield ΔAR A2  7 g/L 0.0% 10.8% A3*  37 g/L 0.5% 10.8% A4 67 g/L 6.7% 9.7% A5  97 g/L 16.7% 8.9% A6 127 g/L 26.9% 8.4% B1 157 g/L37.0% 7.7% B2 187 g/L 47.1% 7.1% B3 217 g/L 57.4% 6.4% B4 247 g/L 67.8%5.8% B5 277 g/L 78.0% 5.3% B6 300 g/L 84.4% 5.0% B7 Wash 87.0% 4.8% C1Wash 88.5% 4.7% C2 Wash 89.6% 4.6% *Dynamic Binding Capacity (DBC) = 39g/L

6.1.3. Example AEX 2 Demonstration of AR Reduction with AEX Adsorbents

This data set is compiled to demonstrate the AR reduction achieved withthree different AEX adsorbents. Each resin was evaluated usingadalimumab at an acetate concentration determined from the processoutlined in Example 6.1.2. and at pH values below, near, and above thepI (e.g. pH 8.5 to 9.0). Table 5 outlines the results from theseexperiments.

TABLE 5 Effect of AEX Resins on AR reduction of Adalimumab Resin BufferCondition Load Yield ΔAR Poros 50PI 5 mM Acetate/Tris pH 8.5 150 g/L 90%2.4% 5 mM Acetate/Tris pH 8.5 300 g/L 94% 0.9% 5 mM Acetate/Tris pH 8.7150 g/L 87% 3.6% 5 mM Acetate/Tris pH 8.7 300 g/L 94% 1.2% 5 mMAcetate/Tris pH 9.0 150 g/L 83% 3.9% 5 mM Acetate/Tris pH 9.0 300 g/L92% 1.5% Poros 50HQ 18 mM Acetate/Tris pH 8.5 250 g/L 91% 3.8% 18 mMAcetate/Tris pH 8.5 350 g/L 88% 2.2% 18 mM Acetate/Tris pH 8.7 250 g/L85% 6.0% 18 mM Acetate/Tris pH 8.7 350 g/L 84% 3.1% 18 mM Acetate/TrispH 8.9 250 g/L 67% 5.9% 18 mM Acetate/Tris pH 8.9 350 g/L 75% 3.6%CaptoDEAE 10 mM Acetate/Tris pH 8.5 150 g/L 98% 0.7% 10 mM Acetate/TrispH 8.5 300 g/L 97% 0.1% 10 mM Acetate/Tris pH 8.7 150 g/L 78% 7.1% 10 mMAcetate/Tris pH 8.7 300 g/L 95% 2.5% 10 mM Acetate/Tris pH 9.0 150 g/L29% 9.2% 10 mM Acetate/Tris pH 9.0 300 g/L 82% 5.0%

This data set is compiled to demonstrate the AR reduction achieved witheight different AEX adsorbents. Each resin was tested using an advancedscreening method using the process outlined in Example 6.1.2., andsubjected to four runs using adalimumab at two different pH (e.g. pH 8.7and 9.0) and two different acetate concentrations (e.g. 10 mM and 20mM). In these experiments, the instantaneous (e.g. not accumulative) ARreduction was measured by analyzing the load fraction at 150 g/L andsubsequently compared across all resins. Table 6 outlines the resultsfrom these experiments.

TABLE 6 Advanced Screen of AEX Resins for AR reduction of AdalimumabInstantaneous AR Resin pH Acetate Reduction @ 150 g/L Poros 50HQ 8.7 10mM 15.0% 20 mM 10.7% 9.0 10 mM 8.6% 20 mM 13.4% Poros 50PI 8.7 10 mM6.2% 20 mM −0.1% 9.0 10 mM 6.5% 20 mM 3.0% Capto DEAE 8.7 10 mM 9.3% 20mM −0.2% 9.0 10 mM 8.6% 20 mM 7.8% Capto Q Impres 8.7 10 mM 12.3% 20 mM4.2% 9.0 10 mM 12.3% 20 mM 6.5% QAE-550C 8.7 10 mM 10.1% 20 mM 3.5% 9.010 mM 7.8% 20 mM 4.5% DEAE 650M 8.7 10 mM 5.2% 20 mM 0.1% 9.0 10 mM 6.9%20 mM −2.7% GigaCap Q 650M 8.7 10 mM 8.1% 20 mM 5.8% 9.0 10 mM 1.8% 20mM 0.4% TMAE HiCap 8.7 10 mM 4.1% 20 mM 2.8% 9.0 10 mM 1.2% 20 mM −0.1%

This data set is compiled to demonstrate the AR reduction achieved withtwo different AEX chromatographic membranes. Each membrane was testedusing conditions outlined in Table 6. The results from these experimentsare presented in Table 7.

TABLE 7 Effect of AEX Chromatographic Membrane on AR reductionofAdalimumab Chromatographic Membrane Equil/Wash Buffer Load Yield ΔARSartobind STIC 10 mM Acetate/Tris pH 8.7 500 g/L  94% 1.7% 20 mMAcetate/Tris pH 9.0 500 g/L 100% 0.7% Sartobind Q 20 mM Acetate/Tris pH9.0 500 g/L 100% 0.3%

This data set is compiled to demonstrate the AR reduction achieved withtwo different charged depth filters. The results from these experimentsare presented in Table 8.

TABLE 8 Effect of Charged Depth Filters on AR reduction of AdalimumabDepth Filter Media Equil/Wash Buffer Load Yield ΔAR CUNO 18 mMAcetate/Tris pH 8.7 500 g/m² 92% 1.9% BioCap 25 X0HC 18 mM Acetate/TrispH 8.7 500 g/m² 84% 1.1%

6.1.4. Example AEX 3 Demonstration of AR Reduction with OtherAntibodies, Mab B And Mab C

AR reduction technology of the current invention has been demonstratedwith multiple antibodies using AEX adsorbents. Antibodies have differentamount charged residues and at different positions, leading to a chargeinteraction behavior on an AEX column that differs from one antibody toanother, Therefore the impact of anion type, anion concentration isdifferent for each antibody.

Table 9 and Table 10 below show the data for MAB B and MAB C. The dataclearly demonstrates that the AR reduction technology works veryeffectively for other antibodies.

TABLE 9 AR reduction for mAb B, pI ~9.1 Resin Buffer Condition pH LoadYield ΔAR Poros 50PI 5 mM Acetate/Tris 9.5 300 g/L 83% 1.1% 9.1 300 g/L94% 1.6% 8.5 300 g/L 98% <0.5% Poros 50HQ 10 mM Acetate/Tris 9.5 300 g/L69% <0.5% 9.1 300 g/L 78% 5.7% 8.5 300 g/L 81% 3.4% Capto DEAE 10 mMAcetate/Tris 9.5 300 g/L 69% 4.2% 9.1 300 g/L 82% 4.9% 8.5 300 g/L 96%<0.5%

TABLE 10 AR reduction for mAb C, pI ~7.0 Resin Buffer Condition pH LoadYield ΔAR Poros 50PI 12 mM Acetate/Tris 7.5 300 g/L 90% 2.6% 7.0 300 g/L89% 2.2% 6.5 300 g/L 87% 4.0% Poros 50HQ 45 mM Acetate/Tris 7.5 300 g/L86% 1.2% 7.0 300 g/L 88% 1.2% 6.5 300 g/L 91% 0.7% Capto DEAE 25 mMAcetate/Tris 7.5 300 g/L 79% 1.8% 7.0 300 g/L 80% 1.9% 6.5 300 g/L 89%1.8%

6.1.5. Example AEX 4 Demonstration of AR Reduction with Different pHConditions—Adalimumab

The AR species in the current invention is bound during the loadingstep; therefore the binding pH is a key variable. The anionconcentration that provides the desired performance will vary with theoperational pH.

In this example, data compiled from different experiments is shown todemonstrate the impact of the pH choice, relative to the pI of theprotein on AR reduction. This data set provides the basis for oneskilled in the art to determine a pH range to perform the experiments toimplement the current invention. Furthermore, this reiterates the factthat the pH choice depends on several factors and the relationshipbetween pH and AR reduction is also mAb dependent

In this example, adalimumab and Poros 50PI were chosen. The experimentswere performed at a concentration of 5 mM acetate/arginine at each pHspecified. Adalimumab was prepared at 5 mM acetate/arginine at each pHspecified and loaded to the column at 300 g-protein/L of resin. Thecolumn was washed with 20 CVs of the equilibration buffer. The resultsshowing the pH effect on AR reduction is shown in FIG. 4.

It is also clear that the AR reduction can be achieved with the presentinvention with a range of pH choices in the range of ±0.5 pH units fromthe pI of multiple mAbs, which are listed in Table 11. Each of theseexperiments was performed with Poros50HQ resin at a 300 g/L load with anacetate/Tris buffer system.

TABLE 11 AR reduction at pH above, at, and below protein pI Range pH-pIMolecule Yield AAR pH > pI 0.2 Adalimumab 71% 7.0% 0.5 mAb B 69% 3.4%0.5 mAb C 86% 1.2% pH~pI 0 Adalimumab 86% 5.9% 0 mAb B 78% 5.7% 0 mAb C88% 1.2% pH < pI −0.2 Adalimumab 93% 4.1% −0.5 mAb B 81% <0.5% −0.5 mAbC 91% 0.7%

6.1.6. Example AEX 5 Demonstration of AR Reduction with Different IonConcentrations—Adalimumab

Anion concentration is a key variable in the performance of anionexchange chromatography. For every combination of antibody/resin/pHthere is a range of anion concentrations that provides AR reduction; thestrategy outlined in Example 6.1.2. can be followed to determine the ARreduction and the corresponding recovery for each anion concentration.

Table 12 below shows the effect of anion concentration on AR reduction.The table also includes the effect of anion concentration for differentpH values. The data demonstrates that the AR reduction can beeffectively achieved over a range of anion concentrations at each pH andthat the concentration ranges depend on the pH.

TABLE 12 Effect of Anion Concentration and pH on AR reduction Resin pHBuffer Condition Load Yield ΔAR Poros 50PI 9 5 mM Acetate/Arginine 300g/L 81% 4.8% 10 mM Acetate/Arginine 227 g/L 80% 2.4% 18.5 mMAcetate/Arginine 107 g/L 88% 1.0% 8.8 5 mM Acetate/Arginine 300 g/L 93%4.5% 10 mM Acetate/Arginine 227 g/L 88% 2.5% 18.5 mM Acetate/Arginine108 g/L 96% 1.2%

6.1.7 Example AEX 6 Demonstration of AR Reduction with Different BufferSystems with Adalimumab

The anion type and concentration are key variables in Anion ExchangeChromatography. The invention has been demonstrated with Acetate andFormate as the anion type and Tris and Arginine as the counter cationtype. As one skilled in the art would appreciate the optimal pH andcation concentration is different for each cation type/mixture and wasderived by using the strategy outlined in Example 6.1.2. Table 13 showsthe data of AR reduction and corresponding recovery for the differentanion/cation types.

TABLE 13 Effect of Anion/Cation Type AR reduction Resin Buffer ConditionLoad Yield ΔAR Poros 50PI 5 mM Acetate/Tris, pH 8.7 300 g/L 94% 1.2% 2.5mM Formate/Tris, pH 8.7 300 g/L 92% 1.3% 5 mM Acetate/Arginine, pH 8.8300 g/L 93% 4.5% Poros 50HQ 15 mM Acetate/Arginine, pH 8.7 300 g/L 89%3.2% 10 mM Formate/Tris, pH 8.7 300 g/L 83% 4.9% 18 mM Acetate/Tris, pH8.7 300 g/L 86% 5.9% Capto DEAE 10 mM Acetate/Tris, pH 8.7 300 g/L 95%2.5% 10 mM Formate/Tris, pH 8.7 300 g/L 94% 1.0% 5 mM Acetate/Arginine,pH 9.0 200 g/L 41% 7.5%

6.1.8. Example AEX 7 Demonstration of Ar Reduction with DifferentLoading

Furthermore, the strategy outlined in Example 6.1.2. to reduce acidicspecies through careful control of buffer anion type, anionconcentration, AEX adsorbent, and pH can be applied to any range ofprotein loading. A range of relevant protein loadings (e.g. 100-350 g/L)for Poros 50HQ at pH 8.7 using Acetate as the anion is shown in Table14, displaying a robust AR reduction across the loading rangeinvestigated.

TABLE 14 Impact of Column loading Yield Load (100-100 mAU) ΔAR 100 g/L78% 9.7% 200 g/L 78% 4.7% 250 g/L 85% 6.0% 300 g/L 89% 3.9% 350 g/L 84%3.1%

6.1.9, Example AEX 8 Demonstration of Ar Reduction with Different LoadConcentration

Furthermore, the strategy outlined in Example 6.1.2. to reduce acidicspecies through careful control of buffer anion type, anionconcentration, AEX adsorbent, and pH can be applied to any range ofcolumn feed streams of varying protein concentration. A range of varyingprotein load concentration for a 300 g/L load of adalimumab to Poros50HQ at 15 mM acetate/Tris pH 8.7 is shown in Table 15.

TABLE 15 Effect of Protein Load concentration Load Yield Concentration(100-100 mAU) ΔAR  5 mg/mL 90% 4.7% 10 mg/mL 86% 4.5% 15 mg/mL 85% 6.3%20 mg/mL 84% 6.2%

6.2.10. Example AEX 9 Alternative Wash Modalities

In this example, adalimumab and Poros50HQ resin were selected. In eachexperiment, variations were made in the equilibration, loading, andwashing pH values at a given acetate concentration (as specified). Table16 and Table 17 show the effect of the pH variation in the step yieldand AR reduction.

TABLE 16 Differences in pH in Equil/Wash/Load Poros 50HQ - 15 mMAcetate/Tris - pH 8.7 - 200 g/L Equilibration Yield pH Load pH Wash pH(100-100 mAU) ΔAR 8.7 8.7 8.5 83% 8.7% 9 8.5 8.5 89% 5.1% 9 100 g/L atpH 9.0 8.5 94% 4.5% 100 g/L at pH 8.5

TABLE 17 Differences in pH in Load/Wash Poros 50HQ - 18 mM Acetate/TrispH 8.7 Load pH Wash pH Load Yield ΔAR 8.6 8.4  75 g/L 88.8% 4.1% 8.6 8.5125 g/L 89.5% 4.2% 8.6 8.6 100 g/L 75.5% 5.3% 8.7 8.4 100 g/L 93.8% 4.1%8.7 8.5 100 g/L 81.7% 3.5% 8.7 8.5  75 g/L 94.5% 4.0% 8.7 8.6 125 g/L81.1% 5.4% 8.7 8.6  75 g/L 65.8% 6.5% 8.8 8.4 125 g/L 93.5% 3.8% 8.8 8.5100 g/L 83.7% 5.8% 8.8 8.6 100 g/L 78.4% 6.4% 8.8 8.6  75 g/L 72.7% 7.0%As discussed in the previous sections, the operational pH and itsrelation to the product pI is important in the reduction of AR speciesin AEX. Similarly, the operational pH relative to the pKa of the AEXadsorbent is also important as many mAbs have pT similar to the pKa ofthe AEX adsorbent. This effect is shown in FIG. 24 for mAb B withseveral different AEX adsorbents, with different pKa values, run at withan acetate/Tris buffer at pH 9.1.

As described in previous sections the Acidic Region for Adalimumab isfurther grouped into two regions termed AR1 and AR2, based on a certainretention time of the peaks seen on the WCX-10 method. Thecharacteristics of the variants in these two regions are expected to bedifferent and hence the methods that reduce variants belonging to thesegroups can be specifically delineated.

Further, in addition to achieving a certain AR reduction, it may bedesirable to achieve a certain absolute level of AR levels, inconsideration of reducing or removing certain variants. The capabilityof the current invention in achieving a certain absolute level of AR,AR1 and AR2 is demonstrated in Table 18. The method of the currentinvention can effectively reduce AR2 levels, as an overall decrease inAR levels is achieved. The method can be used to achieve a targetabsolute level, as exemplified by the data presented in Table 18.Multiple species are present under the group of AR2 and that the currentmethod of invention can be used to reduce such sub-species. The methodof the current invention can effectively achieve AR reduction as well asachieve a target absolute level of acidic species as exemplified by thedata presented in Table 18.

TABLE 18 AR1, AR2, and AR removal Buffer Final Final Resin Condition pHLoad Yield ΔAR1 AR1 ΔAR2 AR2 ΔAR Poros 5 mM 8.5 150 g/L 90% 0.7% 1.5%1.7% 9.4% 2.4% 50PI Acetate/Tris 300 g/L 94% 0.3% 1.9% 0.6% 10.5% 0.9%8.7 150 g/L 87% 0.9% 1.2% 2.7% 8.2% 3.6% 300 g/L 94% 0.4% 1.7% 0.8%10.1% 1.2% 8.9 150 g/L 83% 1.1% 1.4% 2.8% 8.4% 3.9% 300 g/L 92% 0.7%1.8% 0.7% 10.5% 1.5% Poros 18 mM 8.5 250 g/L 91% 2.9% 1.1% 0.9% 10.8%3.8% 50HQ Acetate/Tris 350 g/L 88% 2.7% 1.3% −0.5% 12.2% 2.2% 8.7 250g/L 88% 3.1% 0.9% 2.9% 9.0% 6.0% 350 g/L 84% 2.8% 1.2% 0.3% 11.6% 3.1%8.9 250 g/L 67% 2.6% 1.4% 3.2% 8.6% 5.9% 350 g/L 75% 2.3% 1.7% 1.3%10.5% 3.6% CaptoDEAE 10 mM 8.5 150 g/L 98% −0.1% 2.1% 0.8% 10.0% 0.7%Acetate/Tris 300 g/L 97% 0.0% 2.0% 0.1% 10.8% 0.1% 8.7 150 g/L 78% 2.4%0.8% 4.7% 6.4% 7.1% 300 g/L 95% 1.5% 1.7% 1.0% 10.1% 2.5% 8.9 150 g/L29% 2.1% 0.8% 8.0% 3.0% 10.2% 300 g/L 82% 1.7% 1.2% 3.3% 7.7% 5.0%

6.1.11. Example AEX 10 Demonstration of HCP and Aggregate Reduction inAddition to AR Reduction

AEX chromatography is effective in reducing aggregate and HCP levels. Inthe present invention, it has been demonstrated that HCP and aggregatelevels can be effectively reduced under operating conditions selectedfor AR reduction. Table 19 and Table 20 shows the aggregate and HCPremoval achieved along with AR reduction. The data clearly shows thatother process related and product related substances/impurities can beachieved using the current invention on the AEX adsorbents, and hencefunctions as an effective polishing step in the large scale purificationof monoclonal antibodies.

TABLE 19 Aggregate removal during AEX Chromatography ΔAggregate BufferCondition Load Yield Absolute Relative ΔAR 5 mM Acetate/Tris, 300 g/L81% 0.92% 93% 4.5% pH 9.0 10 mM Acetate/Tris, 227 g/L 80% 0.81% 88% 2.4%pH 9.0 18.5 mM Acetate/Tris, 107 g/L 88% 0.37% 41% 1.0% pH 9.0 5 mMAcetate/Tris, 300 g/L 93% 0.91% 91% 4.5% pH 8.8 10 mM Acetate/Arginine,227 g/L 88% 0.67% 77% 2.5% pH 8.8 18.5 mM Acetate/Arginine, 108 g/L 96%0.34% 40% 1.2% pH 8.8

TABLE 20 HCP Removal during AEX Chromatography Poros 50PI - D2E7 - 300g/L Load HCP Pool HCP HCP Buffer Condition Yield (ng/mL) (ng/mL) (LRF)ΔAR 5 mM Acetate/Tris, 81% 11,617 69 2.2 4.8% pH 9.0 10 mM;Acetate/Tris, 95% 83 2.1 0.8% pH 9.0 5 mM Acetate/Tris, 93% 13,507 512.4 4.5% pH 8.8 10 mM Acetate/Arginine, 97% 84 2.2 1.5% pH 8.8

6.1.12. Example AEX 11 Demonstration of Means of Controlling ARReduction

Controlling the final product quality by modifying the process based onthe quality of the intermediate material is an approach that has beenproposed as an effective way of ensuring product quality, with the viewof ensuring safety and efficacy.

Considering that the AR levels generated during cell culture and otherupstream steps can be variable, it is desirable to design a downstreamprocess step that implements a means of controlling the product quality;and to further have a specific means of controlling a process parameterto influence the quality of the product.

In the current invention, such a control is possible, as the pH and load(i.e. g/L) are parameters that can be modified to achieve a desiredseparation of the AR species. For example, to achieve a higher level ofAR reduction at a given anion concentration and pH, the load to thecolumn can be reduced. Additionally, for a given anion concentration andloading, the pH can be increased in order to achieve a higher reductionin AR species.

As an example, and not to be restrictive in any manner, it has beendemonstrated in this example that the AR levels can be controlled bychanging the pH of the load and wash solutions as well as the total loadto the column. A pilot scale Poros HQ column (10 cm diameter×22.5 cmheight, 1.8 L), was used for this study.

The load material and the stock buffer are both prepared at 18 mMAcetate/Tris the specified pH by titrating the affinity capturedmaterial with a stock Tris solution. The AR level of the load materialwas the same for both runs. This experiment demonstrates how the finalAR level can be modulated, while maintaining acceptable yields, byadjusting the pH and protein load to the column, shown in Table 21.

TABLE 21 Modulating AR Reduction using Process Analytical Technologyapproach Final Buffer Condition Load Yield ΔAR AR 18 mM Acetate/Tris, pH8.7 200 g/L 77% 5.6% 5.5% 18 mM Acetate/Tris, pH 8.5 300 g/L 89% 3.1%8.2%

6.2. Cation Exchange Chromatography Examples 6.2.1. Example CEX 1Determining Operating Conditions Appropriate for A Mab: Resin: BufferCombination

The demonstration of the current invention for a specific antibody &resin is provided in this example, and consists of

-   -   1. Choosing a pH that is below the pI of the protein.    -   2. Choosing a NaCl concentration in the range of 100 to 150 mM        and performing the experiments at, for example, 115, 125, 135        concentrations.    -   3. Determining the acidic species distribution in the ft/wash        fraction vs the elution.    -   4. Choosing a NaCl concentration that provides the desired        acidic species levels and recovery

In this example, adalimumab was chosen and Poros XS was chosen. Theexperiments were performed at pH 6.0. The process chromatograms areshown in FIG. 5. The recovery vs AR reduction curves for each of theexperiments is shown in FIG. 6 and Table 22. From this set ofexperiments, a sodium concentration of 125 mM can be chosen and suchthat the recovery of the eluate is 74%, which provides an AR reductionof 5.4%. Alternately, an AR reduction value of 5.4% can be chosen whichwill provide a recovery of ˜75%.

This general approach is used to determine the appropriate operatingcondition for any resin/mAb combination, to implement the invention.

In practicing certain embodiments of the current invention, the acidicspecies reduction desired can be achieved by appropriate pooling of theelution fraction with the wash fractions. In the example described inthe previous section the elution fractions can be pooled with washfractions as shown in Table 22 to achieve AR reductions from about 1percent to 7 percent depending on the fractions pooled. This approachcan be implemented to achieve a target yield and AR reduction asexemplified in FIG. 6.

TABLE 22 Wash fractions and eluate combination versus AR reductionRecovery % AR Wash Fractions (%) Reduction Eluate 74 5.4 Eluate +Fraction 1 82 4.3 Eluate + Fraction 1 + Fraction 2 88 3.0 Eluate +Fraction 1 + Fraction 2 + Fraction 3 95 0.9 Eluate + Fraction 1 +Fraction 2 + Fraction 3 + 96 0.1 Fraction 4

6.2.2. Example CEX 2 Demonstration of AR reduction with CEX Adsorbents

This data set is compiled to demonstrate the AR reduction achieved with8 different CEX adsorbents. Conditions were derived for each resin basedon the strategy outlined in Example 6.2.1. Table 23 outlines theconditions used and the AR reduction achieved and the correspondingrecovery achieved.

The data clearly shows that the technology is robust in delivering ARreduction in all the 10 resins. As described in Example 6.2.1., the ARreduction can be balanced with recovery and an optimal condition can bechosen. Experiments were performed at pH 7.5. 29 mM Tris-acetate wasused for pH control.

TABLE 23 Effect of CEX adsorbents on AR reduction Tris concentrationYield % AR Resin (mM) (%) Reduction Poros XS 135 103.3 0.7 140 78.6 6.8145 72.6 7.3 Poros HS 100 70.0 6.7 105 68.7 7.1 110 60.6 7.6 Capto SPImpRes 50 71.5 5.7 55 61.0 6.3 60 46.2 6.8 Nuvia S 75 67.6 10.0 80 54.310.8 85 41.0 12.2 Giga Cap CM 650 55 70.3 6.0 57.5 62.7 7.0 60 55.6 8.6Eshmuno S 65 52.7 9.0 70 35.4 11.2 75 22.7 12.2 Giga Cap S 650 65 66.38.4 70 43.6 11.1 75 31.4 12.1 CM Hyper D 45 72.2 8.9 47.5 63.2 9.9 5051.5 10.3

6.2.3. Example CEX 3 Demonstration of AR Reduction with OtherAntibodies: mAb B And mAb C

AR reduction technology of the current invention has been demonstratedwith multiple antibodies using CEX Adsorbents. Antibodies have differentamounts of charged residues and at different positions, leading to acharge interaction behavior on a CEX column that differs from oneantibody to another. Therefore the impact of cation type, cationconcentration is different for each antibody.

For each antibody/resin combination, the experimental strategy outlinedin Example 6.2.1. was employed to determine the cation concentration foreach cation type that provided AR reduction.

Table 24 and Table 25 below shows the data for MAB B and MAB C. The dataclearly demonstrates that the AR reduction technology works veryeffectively for other antibodies. It is also clear that theconcentration ranges are different between different antibodies. The pHrange chosen was related to the isoelectric point of the antibody andwas chosen to be approximately 1 to 2 units less than the pI of themolecule.

TABLE 24 AR reduction for molecule B Buffer Concentration Yield % ARResin System (mM) pH (%) Reduction Poros XS Tris Acetate 120 7.5 57.28.4 125 46.5 9.3 130 37.1 10.3 Nuvia S 85 72.5 16.6 90 56.1 16.9 95 44.217 CM Hyper D 50 73 8.2 55 62 9.2 60 52.6 9.2

TABLE 25 AR reduction for molecule C Buffer Concentration Yield Load %AR Resin System (mM) pH (%) % AR Reduction Poros XS Tris 40 6.0 87.415.6 8.5 Acetate 45 56.8 15.7 12.8 50 31.3 15.7 14.3 Nuvia S 35 45.111.5 11.2 37 28.5 15.4 15.2 40 15.3 15.2 15.2 CM Hyper 18 83.6 16.3 6.3D 20 64.9 16.3 11.2 22 50.7 16.4 12.3

6.2.4. Example CEX 4 Demonstration of AR Reduction with Different pHConditions—Adalimumab

The AR species in the current invention is removed in the Flowthrough/Wash fraction. Therefore the binding pH is a key variable. Thecation concentration that provides the desired performance will varywith the binding pH. Therefore for each binding pH, the experimentalstrategy outlined in Example 6.2.1. is carried out to determine therange of ion concentration that results in AR reduction.

The results of the experiments with different pHs for Adalimumab isshown in Table 26. As can be seen, at lower pH, the cation concentrationrequired to achieve AR removal in the wash fraction is higher. It isunexpected that the AR reduction is significantly more robust andoptimal at higher pHs (closer to pI) than at lower pHs. It is notobvious to one skilled in the art to operate a cation exchangechromatography at pH closer to pI as shown in Table 27. Literature datasuggests an optimal pH of at least 3 units less than the pI of themolecule.

TABLE 26 Effect of pH on AR reduction Buffer Concentration Yield % AR pHResin Buffer System (mM) (%) Reduction 5.5 Poros XS Tris Acetate 35058.2 5.9 6.5 225 61.4 6.4 7 170 75.3 5.6 7.5 140 78.6 6.8 8 125 75.8 5.77.5 CM Hyper Ammonium Sulfate 4 77.9 7.4 6 D Sodium Chloride 45 86.1 46.8 30 71.5 7 7.5 10 71.3 6.8 7.5 Tris Acetate 45 72.2 8.9

TABLE 27 Effect of delta pH and pI on AR reduction % AR pI- Buffer[Cation] Yield Reduc- pH Molecule Resin system (mM) (%) tion 1.1Adalimumab Poros XS Arginine/ 60/29 58.9 7.8 Tris Acetate 2.2 Sodium 12573.5 5.4 1.8 Chloride 75 90 1.5 1.1 50 72.1 7.2 3.1 Tris Acetate 35058.2 5.9 2.1 225 61.4 6.4 1.6 170 75.3 5.6 1.1 145 72.6 7.3 0.6 125 75.85.7 1.6 mAb B Poros XS Tris Acetate 120 57.2 8.4 1.6 CM Hyper D TrisAcetate 50 73 8.2 1.6 Nuvia S Tris Acetate 85 72.5 8.4 1.0 mAb C PorosXS Tris Acetate 40 87.4 8.5 1.0 CM Hyper D Tris Acetate 18 83.6 6.3 1.0Nuvia S Tris Acetate 35 45.1 11.2

6.2.5. Example CEX 5 Demonstration of AR Reduction with Different IonConcentrations—Adalimumab

Cation concentration is a key variable in the performance of cationexchange chromatography. For every combination of antibody/resin/pHthere is a range of cation concentrations that provides AR reduction;the strategy outlined in Example 6.2.1 can be followed to determine theAR reduction and the corresponding recovery for each cationconcentration.

Table 28 below shows the effect of cation concentration on AR reduction.The table also includes the effect of cation concentration for differentpH values. The data demonstrates that the AR reduction can beeffectively achieved over a range of cation concentrations at each pHand that the concentration ranges depend on the pH. The table alsoincludes an example of the concentration range for a different cationtype.

TABLE 28 Effect of cation concentration and pH on AR reduction Cationconcentration Yield % AR (mM) Buffer system pH Resin (%) Reduction 60/29Arginine/Tris 7.5 Poros XS 58.9 7.8 65/29 Acetae 47.4 8.7 23 80.5 5.8 2572.9 7.3 27 52.2 9.5 115 Sodium Chloride 6 85.4 4.2 125 73.5 5.4 13048.7 7.1 75 68 90 1.5 90 53.7 2.1 45 7.5 60.7 7.9 50 72.1 7.2 350 TrisAcetate 5.5 58.2 5.9 375 38.4 7.4 400 29.9 6.2 225 6.5 61.4 6.4 250 59.56.6 275 37.6 7.8 300 21.6 8.8 165 7 83.8 4.3 170 75.3 5.6 175 70.3 5.7140 7.5 78.6 6.8 145 72.6 7.3 150 69.2 7.8 175 29.8 10.3 125 8 75.8 5.7130 67.7 6.5 135 57.4 7.5

6.2.6 Example CEX 6 Demonstration of Ar Reduction with Different BufferSystems with Adalimumab

The cation type and concentration are key variables in Cation ExchangeChromatography. The invention has been demonstrated with Tris,Sodium/Tris, Ammonium/Tris and Arginine/Tris as cation types/mixtureswith effective reduction of AR in each case. As one skilled in the artwould appreciate the optimal pH and cation concentration is differentfor each cation type/mixture and was derived by using the strategyoutlined in Example 6.2.1. Experiment were performed at pH 7.5. 29 mMTris-acetate was used for pH control. Table 29 shows the data of ARreduction and corresponding recovery for the different cationtypes/mixtures.

TABLE 29 Effect of cation types/mixtures on AR reduction Cationconcentration Yield % AR Buffer System Resin (mM) pH (%) ReductionArginine/Tris Poros XS 60 7.5 58.9 7.8 acetate Ammonium 25 72.9 7.3Sulfate Sodium Chloride 50 72.1 7.2 Tris Acetate 140 78.6 6.8 AmmoniumCM Hyper 4 77.9 7.4 Sulfate D Sodium Chloride 10 71.3 6.8 Tris Acetate45 72.2 8.9 Ammonium Nuvia S 11 66.6 12.6 Sulfate Sodium Chloride 2075.9 10.5 Tris Acetate 75 67.6 10

6.2.7. Example CEX 7 Demonstration of AR Reduction with DifferentLoading

Furthermore, the strategy outlined in Example 6.2.1. to reduce acidicspecies through careful control of buffer cation type, concentration andpH can be applied to any range of protein loading which represents anoperational mode of binding followed by elution, i.e. not overloaded ora column load factor below that of the adsorbents binding capacity. Arange of relevant protein loadings for Poros XS at pH 7.5 using Tris asthe cation is shown in Table 30 showing robust AR reduction.

TABLE 30 Impact of Column loading Column Loading (g product/ BufferConcentration % AR L resin) System (mM) pH Yield (%) Reduction 25 Tris160 7.5 83.6 6.4 30 155 79.4 6.0 35 140 87.4 4.8 38 140 83.5 5.0 40 14076.4 6.0 42 140 74.5 5.7 45 140 67.0 6.6

6.2.8. Example CEX 18 Demonstration of AR Reduction with Different LoadConcentration

Furthermore, the strategy outlined in Example 6.2.1. to reduce acidicspecies through careful control of buffer cation type, concentration andpH can be applied to any range of column feed streams of varying proteinconcentration. A range of varying protein load concentration for PorosXS at pH 7.5 using Tris as the cation is shown in Table 31 showingrobust AR reduction.

TABLE 31 Effect of Protein Load concentration Load Concen- % ARConcentration Buffer tration Yield Reduc- (mg/mL) Resin System (mM) pH(%) tion 3 Poros XS Tris 140 7.5 77.3 7 4 Acetate 145 60.7 7 5 140 78.76.7 5 145 64.1 7 6 145 59.5 6.9 7 140 77.6 6.5

As described above, the Acidic Region for Adalimumab is further groupedinto two regions termed AR1 and AR2, based on a certain retention timeof the peaks seen on the WCX-10 method. The characteristics of thevariants in these two regions are expected to be different and hence themethods that reduce variants belonging to these groups can bespecifically delineated.

Further, in addition to achieving a certain AR reduction, it may bedesirable to achieve a certain absolute level of AR levels, inconsideration of reducing or removing certain variants. The capabilityof the current invention in achieving a certain absolute level of AR,AR1 and AR2 is demonstrated in Table 32.

The specific species comprising the AR1 species can be identified andquantitated, to demonstrate reduction of such species by methods of thecurrent invention. Two of such species, Glycated mAb, and MGO modifiedmAb have been identified and shown to be reduced by the methods of thisinvention. While these species are among the Acidic Species part of thecharge variants, the acidic species typically described in theliterature is the deamidated mAb, which is distinctly different.

TABLE 32 The final impurity level Cation Conc. Yield % Final % FinalBuffer System (mM) pH (%) AR1 AR Arginine/Tris 60 7.5 58.9 0.3 5.8Acetate 65 7.5 47.4 0.3 4.7 Ammonium Sulfate 23 7.5 80.5 0.6 8.3 25 7.572.9 0 6.4 27 7.5 52.2 0.4 5.0 Sodium Chloride 115 6 85.4 1.3 10.2 125 673.5 0 8.1 135 6 48.7 0 6.1 75 6.8 90 1.4 10.9 90 6.8 53.7 0.7 11.2 457.5 60.7 0 6.2 50 7.5 72.1 0 7.8 Tris Acetate 350 5.5 58.2 0 7.7 375 5.538.4 0.1 6.2 400 5.5 29.9 1.5 7.3 225 6.5 61.4 0.8 7.2 250 6.5 59.5 06.8 275 6.5 37.6 0 5.6 300 6.5 21.6 0 4.7

The method of the current invention can effectively reduce AR2 levels,as an overall decrease in AR levels is achieved. The method can be usedto achieve a target absolute level, as exemplified by the data presentedin Table 32.

The method of the current invention can effectively achieve AR reductionas well as achieve a target absolute level of acidic species asexemplified by the data presented in Table 32.

6.2.9. Example CEX 9 Demonstration of Glycated and MethylglyoxylatedSpecies Reduction

The strategy outlined in Example 6.2.1. to reduce acidic species throughcareful control of buffer cation type, concentration and pH can befurther extended to specific post-translational modifications. Whileacidic species are defined in the application as impurities that areless retained than the main peak on an analytical weak cation exchange(WCX) HPLC column, specific known product related substances derivedfrom cellular metabolism modification such as glycation andmethylglyoxal (MGO) can be specifically identified as being part of theacidic species. FIG. 7 and FIG. 8 shows the outcome of in-vitro labelingexperiments which demonstrate that glycation and MGO modified antibodyare unique species that are resolved by the WCX method in the AR1 regionof the chromatogram and can be enriched in vitro. Furthermore, theinvention described here shows that glycated and MGO modified antibodycan be effectively removed through the careful control of buffer cationtype, concentration and pH using the CEX as described in Example 6.2.1.Quantitative reduction of AR1 and hence the Glycated and MGO species byCEX and CEX-Mixed Mode resins is show in Table 33 and Table 34.

TABLE 33 Glycated species removal % ARI % AR Buffer Conc. Yield Load %Load % Reduc- Reduc- Resin System (mM) pH (%) AR1 AR tion tion PorosTris 135 7.5 54.0 40.8 58.6 30.8 34.8 XS

TABLE 34 MGO peak removal Concen- % AR1 % AR Buffer tration Reduc-Reduc- Resin System (mM) pH Yield (%) tion tion Toyo Pearl MX Tris 807.5 66.7 2.8 7.2 TRP 650M Poros XS 145 64.1 2.7 7 Nuvia S 90 48.5 3.19.6

6.2.10 Example CEX 10 Demonstration of Lysine Distribution Modification

The strategy outlined in Example 6.2.1. to reduce acidic species alsocan be used to modulate the distribution of C-terminal Lys variants ofmonoclonal antibodies, a known post-translational modification leadingto charge heterogeneity. Some minor changes in the distribution of Lysisoforms is expected through the reduction of acidic species as the WCXanalysis is a compositional analysis. However, through careful controlof buffer cation type, concentration and pH care, in addition toreducing acidic species, the elution pool can be enriched for the morebasic isoforms (Lys 1 and Lys2). Table 35 and FIG. 9 depicts anon-limited example of the impact of pH and cation (Tris) concentrationon basic isoform enrichment.

TABLE 35 Change in Lysine distribution during CEX Chromatography -impact of Tris concentration Buffer % LYS0 % LYS1 % LYS2 BufferConcentration decrease Increase Increase System (mM) pH 1.6 4.4 2.7 3505.5 5 6.5 5.5 375 9.7 7.5 11.9 400 1.9 5 2.9 225 1.9 5.3 3 250 6.1 7.4 6275 11.8 3.6 10.8 300 0.2 5.2 1.6 140 7.5 0.6 5.7 1.8 145 1.8 6.8 2.4150 16.4 14.9 10.3 175

6.2.11. Example CEX 11 Demonstration of HCP and Aggregate Reduction inAddition to AR Reduction

In the present invention, it has been demonstrated that HCP andaggregate levels can be effectively reduced by appropriate adjustment ofthe elution conditions, after washing off the AR enriched species in theflow through/wash fractions.

Table 36 and Table 37 shows the HCP and aggregate removal achieved alongwith AR reduction. The data clearly shows that other process related andproduct related substances/impurities can be achieved using the currentinvention on the CEX adsorbents, and hence functions as an effectivepolishing step in the large scale purification of monoclonal antibodies.

TABLE 36 Aggregate removal during CEX Chromatography % Aggregate %Fragment % Monomer Resin Molecule Buffer system pH Reduction ReductionIncrease CM Hyper D adalimumab 5 mM 7.5 0.04 0.17 0.2 Ammonium Sulfate45 mM Tris 0.01 0.18 0.19 Acetate Nuvia S 11.5 mM 0.16 0.17 0.33Ammonium Sulfate 75 mM Tris 0.09 0.11 0.2 Acetate 22.5 mM 0.08 0.19 0.27Sodium Chloride Poros XS 27 mM 0.75 0.27 1.02 Ammonium Sulfate 140 mMTris 0.51 0.41 0.92 Acetate 145 mM Tris 0.58 0.41 0.98 Acetate Nuvia SmAb B 85 mM Tris 0.19 0.27 0.47 Acetate Poros XS 130 mM Tris 0.36 0.040.39 Acetate Nuvia S mAb C 35 mM Tris 6.0 0.07 0.01 0.07 Acetate PorosXS 50 mM Tris 0.27 0 0.28 Acetate

TABLE 37 HCP Removal during CEX Chromatography Eluate Pool Buffer LoadHCP HCP Reduction Resin Molecule system pH (ng/mg) (ng/mg) fold CM HyperD adalimumab 5 mM 7.5 8105 3844 2.1 Ammonium Sulfate 45 mM Tris 86285615 1.5 Nuvia S 11.5 mM 5314 2405 2.2 Ammonium Sulfate 75 mM Tris 1731712845 1.4 Acetate 22.5 mM 9091 4115 2.2 Sodium Chloride Poros XS 27 mM21857 12574 1.0 Ammonium Sulfate 140 mM Tris 14732 9181 1.7 Acetate 145mM Tris 15359 10113 1.6 Acetate Nuvia S mAb B 85 mM Tris 735 319 2.3Acetate Poros XS 130 mM Tris 2183 404 5.4 Acetate Nuvia S mAb C 35 mMTris 6.0 27 31 0.9 Acetate Poros XS 50 mM Tris 25 15 1.7 Acetate

6.2.11. Example CEX 12 Demonstration of Means of Controlling ARReduction

Controlling the final product quality by modifying the process based onthe quality of the intermediate material is an approach that has beenproposed as an effective way of ensuring product quality, with the viewof ensuring safety and efficacy.

Considering that the AR levels generated during cell culture and otherupstream steps can be variable, it is desirable to design a downstreamprocess step that implements a means of controlling the product quality;and to further have a specific means of controlling a process parameterto influence the quality of the product.

In the current invention, such a control is possible, as the cationconcentration is a single parameter that can be modified to achieve adesired separation of the AR species. For example, to achieve a higherlevel of AR reduction, the Tris concentration of the loading materialand the wash buffer can be decreased, such that the AR enriched speciesis collected in the flow through fraction.

As an example, and not to be restrictive in any manner, it has beendemonstrated in this example that the AR levels can be controlled bychanging the Tris concentration of the load and wash solutions. A pilotscale Poros XS column (10 cm diameter×22 cm height, 1.7 L), was used forthis study.

The load material and the stock buffer are both prepared at 300 mM Trisconcentration at the same pH. The AR level of the load material wasmeasured to be X %. The load material and equilibration/wash buffer arein-line diluted to the target Tris concentration based on predeterminedcorrelation between the AR levels and Tris concentration. Asdemonstrated in the example, when the Tris concentration was adjusted to156 mM, a final AR reduction of 4.1% was achieved, whereas when the Trisconc. was adjusted to 150 mM, a final AR level of 3.1 was achieved etc(Table 38). This allows very predictable control of the AR levelsensuring achievement of the desired product quality.

TABLE 38 Controlling AR Reduction using Process Analytical Technologyapproach Tris conc (mM) Yield (%) % AR Reduction 156 51.9 4.1 150 70.53.1 131 95.3 1.3

In addition to the acidic species reduction demonstrated in Example CEX1 through careful control of the pH cation type and concentration in theload (process stream) and equilibration/wash buffers, the composition ofthe elution buffer can also be used to further improve the productquality profiles. The impact of various cation types, concentration andpH were tested for eluting the product. There is a wide selection forelution buffer as shown in Table 39. The experiments were performedusing Poros XS resin

TABLE 39 Elution buffer types on aggregates removal % Aggregate BufferSystem pH Yield (%) Reduction 200 mM Sodium Sulfate/29 mM Tris 5.2 76.10.36 Acetate 160 mM Sodium Sulfate/29 mM Tris 5.2 82.3 0.82 Acetate 150MSodium Sulfate/29 mM Tris 5.2 78.8 0.90 Acetate 140M Sodium Sulfate/29mM Tris 5.2 78.2 1.00 Acetate 400 mM Sodium Sulfate/29 mM Tris 4.0 78.50.98 Acetate 100 mM Sodium Sulfate/140 mM Tris 5.2 70.9 1.25 Acetate 150mM Sodium Sulfate/140 mM Tris 5.2 79.6 1.05 Acetate 140M SodiumSulfate/140 mM Tris 5.2 75.4 1.07 Acetate 130 mM Sodium Sulfate/140 mMTris 5.2 78.2 1.07 Acetate 300 mM Sodium Sulfate/30 mM Tris 4.6 80.30.57 Acetate 150 mM Sodium Sulfate/29 mM Tris 7.5 75.0 0.92 Acetate

6.2.13. Example CEX 13 Demonstration of Ar Reduction with Cation-HicMixed Mode Resin

The strategy outlined in Example 6.2.1. to reduce acidic species throughcareful control of buffer cation type, concentration and pH can beexpanded to include other chromatography adsorbents such as mixed modeor multi-modal absorbents which include a cation exchange mechanism.Table 40 outlines the conditions used and the AR reduction achieved fortwo cation-hydrophobic interaction mixed mode resins. The data clearlyshows that the technology outlined in Example 6.2.1. is robust indelivering AR reduction for these types of resins across in addition totraditional cation exchange adsorbents. As described in Example 6.2.1,the AR reduction can be balanced with recovery and an optimal conditioncan be chosen. As a further demonstration, molecule 2 was also evaluated(Table 41) with the same outcome showing the same relationship betweencation concentration, recovery and AR reduction. As previously shown inExample 6.2.6, the optimal condition for different molecules varies.Furthermore, this technology when applied to CEX-HIC mixed mode resinsalso shows reduction of impurities as previously described.

TABLE 40 Adalimumab AR Reduction by Cation Exchange Mixed ModeChromatography Tris Buffer Concentration Yield % AR Resin System (mM) pH(%) Reduction Nuvia C Prime Tris 70 7.5 63.8 6.5 Acetate 72.5 7.5 61.16.0 75 7.5 57.1 6.7 Toyo Pearl MX 75 7.5 80 5.7 Trp 650M 80 7.5 66.7 7.285 7.5 51.8 8.6

TABLE 41 Molecule B AR Reduction by Cation Exchange Mixed ModeChromatography Buffer Concentration Yield % AR Resin System (mM) pH (%)Reduction Nuvia C Prime Tris 75 7.5 86.0 2.0 Acetate 85 7.5 74.6 5.9 957.5 61.3 6.8 Toyo Pearl MX 90 7.5 81.1 6.4 Trp 650M 95 7.5 68.8 8.8 1007.5 53.5 10.7

As described in previous sections, the Acidic Region for Adalimumab isfurther grouped into two regions termed AR11 and AR2, based on a certainretention time of the peaks seen on the WCX-10 method. Thecharacteristics of the variants in these two regions are expected to bedifferent and hence the methods that reduce variants belonging to thesegroups can be specifically delineated.

Further, in addition to achieving a certain AR reduction, it may bedesirable to achieve a certain absolute level of AR levels, inconsideration of reducing or removing certain variants. The capabilityof the current invention in achieving a certain absolute level of AR,AR1 and AR2 is demonstrated in Table 42A with Tables 42B and 42Cindicating the levels of additional process-related impurities orproduct-related substances.

The specific species comprising the AR1 species can be identified andquantitated, to demonstrate reduction of such species by methods of thecurrent invention. While these species are among the Acidic Species partof the charge variants, the acidic species typically described in theliterature is the deamidated mAb, which is distinctly different. Theseresults show that the Cation Exchange Resin with additional pendanthydrophobic interaction functionality, is able to provide AR reductioneffectively, similar to the CEX Adsorbents.

TABLE 42A Final acidic species level for Adalimumab Tris BufferConcentration Yield Final Final Final Resin System (mM) pH (%) % AR1 %AR2 % AR Nuvia C Prime Tris Acetate 70 7.5 63.8 0.39 4.64 5.03 72.5 7.561.1 0.36 4.4 4.75 75 7.5 63.8 0.39 4.06 4.45 Toyo Pearl MX 75 7.5 800.6 4.2 4.8 Trp 650M 80 7.5 66.7 0.5 3.2 3.7 85 7.5 51.8 0.2 2.2 2.4

TABLE 42B Aggregates/Fragments Reduction by Cation Exchange Mixed ModeChromatography Buffer % Aggregate % Fragment % Monomer Resin MoleculeSystem pH Reduction Reduction Increase Nuvia C prime adalimumab 70 mMTris 7.5 0.3 0.34 0.63 Toyo Pearl MX Trp 75 mM Tris 0.08 0.56 0.65 650MNuvia C prime Molecule B 85 mM Tris 0.87 1.18 2.04 Toyo Pearl MX Trp 95mM Tris 0.0 1.8 1.8 650M

TABLE 4C HCP Reduction by Cation Exchange Mixed Mode ChromatographyEluate pool Load HCP HCP Fold Resin Compound Buffer pH (ng/mg) (ng/mg)Reduction Toyo Pearl MX Trp 650M adalimumab 70 mM Tris 7.5 202.6 38.95.2 Nuvia C prime 75 mM Tris 205.5 72.8 2.8 Toyo Pearl MX Trp 650MMolecule B 95 mM Tris 983.3 137.1 7.2 Nuvia C prime 85 mM Tris 1011.388.2 11.5

6.2.14. Example CEX 14 Demonstration of Ar Reduction in ProcessCombinations

The method described above for reducing acidic species using cationexchange can be used as an independent operation or in combination withother process steps that provide additional acidic species reduction orthose providing additional complementary and supplementary purification(See tables 43-50). The following process combinations are provided hereas non-limiting examples

1. Affinity→MM→CEX

2. Affinity→AEX→CEX

3. Affinity→CEX

4. CEX Capture→CEX

TABLE 43 AR Reduction by Capto Adhere(mixed mode) followed by Poros XS(CEX) Capto Adhere CEX Cycle B % AR1 % AR Step Yield % % AR1 % ARReduction Reduction MabSure Eluate 2.90 10.08 Viral Inact 89 2.89 10.42Mixed Mode FTW 94 2.26 8.52 0.64 1.90 CEX Load 2.29 8.97 CEX Eluate 910.25 4.88 2.04 4.10 Overall 76 2.65 5.20

TABLE 44 Aggregate reduction by combination of Capto Adhere(mix mode)Poros XS (CEX) Capto Adhere CEX Cycle B % % % % Mono % Agg. % Frag StepYield % Monomer Aggregate Fragment increase decrease decrease MabSure99.08 0.85 0.08 Eluate Viral Inact 89 99.14 0.73 0.13 Mixed Mode 9699.64 0.26 0.10 0.50 0.47 0.03 FTW CEX Load 99.64 0.26 0.10 CEX Eluate89 99.74 0.18 0.08 0.10 0.08 0.02 overall 76 0.66 0.67 0.00

TABLE 45 AR Reduction by Poros PI (AEX) followed by Poros XS (CEX) AEXCEX Cycle C % AR1 % AR Step Yield % % AR1 % AR Reduction ReductionMabSure Eluate 2.90 10.08 AEX Load 2.73 10.16 AEX FTW 90 1.64 6.7 1.093.46 Viral Inact 100 1.39 6.03 CEX Load 2.76 6.18 CEX Eluate 91 0.153.22 2.61 2.96 Overall 82 2.75 6.86

TABLE 46 Aggregate reduction Poros PI (AEX) Poros XS (CEX) AEX CEX CycleC % % % % Mono % Agg. % Frag Step Yield % Monomer Aggregate Fragmentincrease decrease decrease MabSure 99.08 0.85 0.08 Eluate AEX Load 98.671.25 0.03 AEX FTW 90 99.88 0.05 0.07 1.21 1.2 −0.04 Viral Inact 10099.94 0.05 0.02 CEX Load 99.64 0.26 0.10 CEX Eluate 91 99.79 0.13 0.080.14 0.13 0.02 Overall 82 0.71 0.72 0.00

TABLE 47 AR reduction from a Affinity capture pool followed by Poros XS(CEX) % AR1 % AR Step Yield % % AR1 % AR Reduction Reduction MabSureEluate 3.0 10.5 CEX Eluate 82.7 0.3 4.9 2.8 5.6

TABLE 48 Aggregate reduction: Affinity capture pool followed by Poros XS(CEX) % % % % Mono % Agg. % Frag Step Yield % Monomer Aggregate Fragmentincrease decrease decrease MabSure 98.5 1.4 0.1 Eluate CEX Eluate 82.799.7 0.2 0.1 1.2 1.2 0.0

TABLE 49 AR reduction CEX Capture (Fractogel SO3) followed by Poros XS(CEX) 145 mM TA Poros XS D2E7 % AR1 % AR Step Yield % % AR1 % ARReduction Reduction Concentrated 3.3 14.0 Fractogel Eluate VI CEX Eluate72.6 0.44 6.7 2.8 7.3

TABLE 50 Aggregate reduction: CEX Capture (Fractogel) followed by PorosXS (CEX) 145 mM TA Poros XS D2E7 % % % % Mono % Agg. % Frag Step Yield %Monomer Aggregate Fragment increase decrease decrease Concentrated 97.91.5 0.7 Fractogel Eluate VI CEX Eluate 72.6 98.7 1.1 0.2 0.9 0.4 0.5

6.3. Mixed Mode Chromatography Examples 6.3.1. Example M M1 Resin:Buffer Combination

In this example one of the approaches outlined in the generaldescription was employed to determine the operating conditions toimplement the invention. Specifically, a response surface design DOE wasapplied to evaluate mAb AR reductions and recovery yields.

The demonstration of the current invention for a specific antibody &resin is provided in this example, and consists of

-   -   1. Choosing a pH in the range of 6.8 to 8.4.    -   2. Choosing a conductivity in the range of 2.3 to 13.7 mS/cm.    -   3. Determining the acidic species distribution in the ft/wash        fractions.    -   4. Choosing an optimal pH and conductivity that provides the        desired acidic species levels and recovery

In this example, Adalimumab and resin Capto Adhere were chosen. Theexperiments were performed with Tris/Acetate buffer system at target pHand conductivity listed in Table 51 The load material was from Protein Aaffinity capture and pH adjusted. This study demonstrated the effect ofloading pH and conductivity on acidic species reduction. The acidicspecies reduction can be significantly affected by operating pH. ARreduction increased with increasing pH and/or decreasing conductivity(Table 51, Table 52 and FIG. 10)

TABLE 51 DOE study condition Tris Acetate Edge points for Buffer RangeResponse Surface pH 7.0-8.2  6.8, 8.4  Conductivity 4.0-12.0 2.3, 13.7

TABLE 52 DOE Study Operating Conditions and Results Conductivity DOE exppH (mS/cm) ΔAR (%) Yield (%) 1 7.0 4.0 0.4 83 2 7.6 8.0 0.4 73 3 7.6 2.31.3 82 4 7.6 8.0 0.6 68 5 7.6 8.0 0.2 70 6 7.6 8.0 −0.2 69 7 8.2 4.0 2.167 8 7.6 8.0 1.3 69 9 7.0 12.0 −0.2 70 10 7.6 8.0 1.2 71 11 8.2 12.0 1.474 12 6.8 8.0 1.2 76 13 8.4 8.0 1.8 67 14 7.6 8.0 1.4 71 15 7.6 13.7 1.074 16 7.6 8.0 1.6 70 Note: AR reductions and protein recovery yieldswere calculated based on the Flow Through fractions at about loading 200g protein per L of resin.

6.3.2. Example MM 2 Fraction Pooling

In this example, Adalimumab and resin Capto Adhere were chosen. Theexperiments were performed with Tris/Acetate buffer system at pH 7.85and conductivity of 2.5 mS/cm. The load material was from Protein Aaffinity capture and pH adjusted. Column flow through was fractionatedthroughout the entire load and wash phases. Each fraction was analyzedfor acidic species and protein recovery. FIG. 11, FIG. 12 and Table 53demonstrate AR reduction achieved with the corresponding recovery. TheseAR reductions and recoveries correspond to the cumulative pools of thefractions from the start to the various points during the load/wash.This is depicted in Table 53 where the AR reductions corresponding toeach of these pools. This data is plotted in FIG. 11.

TABLE 53 Cumulative AR reduction in Flowthrough/wash fractionsFlowthrough Fraction Yield Δ AR1 Δ AR2 Δ AR ΔLys (Load & wash) (%) (%)(%) (%) (%) A2 23 2.56 3.13 5.69 5.61 A2 + A3 45 2.31 2.19 4.49 4.37A2 + A3 + A4 58 1.83 1.89 3.72 3.63 A2 + A3 + A4 + A5 65 1.57 1.58 3.153.06 A2 + A3 + A4 + A5 + A6 73 1.38 1.32 2.70 2.61 A2 + A3 + A4 + A5 +A6 + B7 86 1.26 1.12 2.38 2.30 A2 + A3 + A4 + A5 + A6 + 89 1.19 0.912.09 2.02 B7 + B6 A2 + A3 + A4 + A5 + A6 + 90 1.14 0.82 1.96 1.89 B7 +B6 + B5 Note: “A” Fractions are load fractions and “B” Fractions arewash fractions

6.3.3. Example M M 3 Demonstration of AR Reduction with Mixed ModeAdsorbents

In this example, Adalimumab was chosen. The experiments were performedwith Tris/Acetate buffer system at pH 7.85 and conductivity of 2.5, 3.5,and 4.5 mS/cm. The same load material was applied to different mixedmode resin columns. The load material was from Protein A affinitycapture and pH adjusted. Table 54 shows that all three mixed mode resinscould reduce mAb acidic species. Due to the differences of resinligands, the AR reduction level may slightly vary under certainconditions.

TABLE 54 Adalimumab AR Reduction and Protein Recovery Yields Processedwith Different Mixed Mode media Tris/Ac Buffer Capto Adhere HEA PPA pH7.85 pH 7.85 pH 7.85 Operating 4.5 3.5 2.5 4.5 3.5 2.5 4.5 3.5 2.5Conditions mS/cm mS/cm mS/cm mS/cm mS/cm mS/cm mS/cm mS/cm mS/cm Yield(%) 50 52 58 49 52 56 40 43 47 AR Reduction 1.8 3.8 3.7 1.1 2.7 3.2 1.42.2 3.5 (%) Yield (%) 68 71 73 65 75 69 61 64 63 AR Reduction 1.1 2.72.7 0.5 1.8 2.1 0.4 1.9 2.6 (%)

6.3.4. Example MM 4 Demonstration of Ar Reduction with Other Antibodies:mAb B And mAb C

In this example, another two different monoclonal antibodies besidesAdalimumab and resin Capto Adhere was chosen. The experiments wereperformed with Tris/Acetate buffer system at multiple pH andconductivity condition. The load materials of all mAbs were from ProteinA affinity capture and pH adjusted. MAB C was also applied to anothertwo MM resins besides Capto Adhere under the same operating conditions.The Table 55 outlines the operating conditions and the AR reductionachieved and the corresponding recovery achieved. The resultsdemonstrate that the technology can also reduce acidic species for othermonoclonal antibodies with optimal pH and conductivity conditions.Experiments were performed with Tris-acetate buffer system.

TABLE 55 AR Reductions and Protein Recovery for different mAb with CaptoAdhere columns conductivity mAb pH (mS/cm) ΔAR (%) Yield (%) D2E7 7.853.5 3.8 52 7.85 2.5 3.7 58 MAB B 6.8 3.0 6.3 51 6.8 4.5 4.2 53 7.0 3.05.1 77 8.0 3.0 3.4 60 MAB C 9.0 3.0 5.3 73 8.5 3.0 3.5 54 8.0 3.0 3.7 50

FIG. 13 displays the MAB B cumulative pool AR broke through the columnof Capto Adhere operated at pH 7.0 and conductivity of 3.0 mS/cm withTris-Acetate buffer. FIG. 14 shows the MAB C cumulative pool AR brokethrough the column of Capto Adhere operated at pH 8.5 and conductivityof 3.0 mS/cm with Tris-Acetate buffer. Both of graphs demonstratesimilar AR breakthrough curves with different AR values comparing toadalimumab (FIG. 12). FIG. 15 presents the AR breakthrough curves of MabC with three different mixed mode resins with Tris-acetate bufferoperated at pH 8.5 and conductivity of 3.0 mS/cm. The data clearlydemonstrates that the AR reduction technology using mixed mode resinsworks very effectively for other antibodies.

6.3.5. Example MM 5 Demonstration of Relative pH on AR Reduction withDifferent Resins Using D2E7 Antibody Material

In this example, data compiled from different experiments is shown todemonstrate the impact of the pH choice, relative to the pI of theprotein on AR reduction. This data set provides the basis for oneskilled in the art to determine a pH range to implement the currentinvention. Further, this reiterates the fact that the pH choice dependson several factors and the relationship between pH and AR reduction isalso mAb dependent. FIG. 16 demonstrates the impact of pH-pI andconductivity on AR reduction which compiled data from the experimentsperformed with Capto Adhere under conditions listed in Table 56. FIG. 17shows the impact of pH-pI and conductivity on MAB B AR reductionincluding the experiments operated with Tris/Acetate buffer system andmultiple mixed mode resins under the conditions listed in Table 57. FIG.18 shows the impact of pH-pI and conductivity on MAB C AR reductionincluding the experiments operated with Tris/Acetate buffer system andmultiple mixed mode resins under the conditions listed in FIG. 16. Allthe load materials were from Protein A affinity capture and pH adjusted.It is also clear that the AR reduction can be achieved with the presentinvention with a range of pH choices, in the range of +0.5 to −2.5 pHunits from pI for D2E7. One skilled in the art can choose an appropriatepH to achieve a target AR reduction.

TABLE 56 Operating conditions and AR reductions for D2E7 ConductivityBuffer system pH pH-pI (mS/cm) AR reduction Tris/Ac 7 −2.02 4 0.4 7.6−1.42 8 0.4 7.6 −1.42 2.3 1.3 7.6 −1.42 8 0.6 7.6 −1.42 8 0.2 7.6 −1.428 −0.2 8.2 −0.82 4 2.1 7.6 −1.42 8 1.3 7 −2.02 12 −0.2 7.6 −1.42 8 1.28.2 −0.82 12 1.4 6.8 −2.27 8 1.2 8.4 −0.57 8 1.8 7.6 −1.42 8 1.4 7.6−1.42 13.7 1.0 7.6 −1.42 8 1.6 7.5 −1.52 3.75 1.7 7.6 −1.42 2.5 2.7 7.6−1.42 2.5 2.0 7.6 −1.42 5 1.3 7.6 −1.42 5 1.1 7.85 −1.17 2 3.5 7.85−1.17 3.75 3.2 7.85 −1.17 3.75 2.1 7.85 −1.17 3.75 2.8 7.85 −1.17 3.752.2 7.85 −1.17 5.5 2.1 8.1 −0.92 2.5 5.0 8.1 −0.92 2.5 2.6 8.1 −0.92 5−0.2 8.1 −0.92 5 −1.1 8.2 −0.82 3.75 2.9 Arg/Ac 8.5 −0.52 1 6.8 9.0−0.02 1 6.5 9.5 0.48 1 1.9 Trol/Ac 7.85 −1.17 1 5.7 8.0 −1.02 1 8.0 8.5−0.52 1 6.0

TABLE 57 Operating conditions and AR reductions for MAB B ConductivitypH pH-pI (mS/cm AR reduction Capto Adhere 6.8 −0.45 3 6.3 7 −0.25 3 6.27.5 0.25 3 4.0 8 0.75 3 3.2 6.8 −0.45 4.5 4.1 7.5 0.25 4.5 3.3 PPA 6.8−0.45 3 1.1 7 −0.25 3 0.9 7.5 0.25 3 1.3 8 0.75 3 0.5 6.8 −0.45 4.5 1.67.5 0.25 4.5 3.0 HEA 6.8 −0.45 3 1.8 7 −0.25 3 1.4 7.5 0.25 3 3.6 8 0.753 0.7 6.8 −0.45 4.5 2.2 7.5 0.25 4.5 0.9

TABLE 58 Operating conditions and AR reductions for MAB C ConductivitypH pH-pI (mS/cm) Δ % AR Capto Adhere 8.0 −1.11 1 1.5 8.5 −0.61 1 3.5 9.0−0.11 1 5.4 PPA 8.0 −1.11 1 −0.4 8.5 −0.61 1 1.1 9.0 −0.11 1 2.1 HEA 8.0−1.11 1 −1.6 8.5 −0.61 1 1.9 9.0 −0.11 1 2.8

6.3.6. Example MM 6 Effect of pH on AR Reduction

Response surface design DOE was applied to evaluate the impact of pH andconductivity on mAb AR reductions. In this example, Adalimumab and CaptoAdhere were chosen. The experiments were performed with Tris/Acetatebuffer system. The load material was from Protein A affinity capture andpH adjusted. Besides the pH and conductivity ranged tested anddemonstrated in Table 59 and Table 60, higher pH ranges were alsostudied (FIG. 19).

The results in FIG. 19 and FIG. 20 demonstrated that mAb acidic speciescan be reduced at wide pH range from 6.8 to 9.5.

TABLE 59 DOE study condition Tris Acetate Edge points for Buffer RangeResponse Surface pH 7.0-8.2  6.8, 8.4  Conductivity 4.0-12.0 2.3, 13.7

TABLE 60 AR reduction and Yield in DOE study Experiment # pHConductivity ΔAR Yield 1 7.0 4.0 0.4 83 2 7.6 8.0 0.4 73 3 7.6 2.3 1.382 4 7.6 8.0 0.6 68 5 7.6 8.0 0.2 70 6 7.6 8.0 −0.2 69 7 8.2 4.0 2.1 678 7.6 8.0 1.3 69 9 7.0 12.0 −0.2 70 10 7.6 8.0 1.2 71 11 8.2 12.0 1.4 7412 6.8 8.0 1.2 76 13 8.4 8.0 1.8 67 14 7.6 8.0 1.4 71 15 7.6 13.7 1.0 7416 7.6 8.0 1.6 70 Note: AR reductions and protein recovery yields werecalculated based on the Flow Through fractions at about loading 200 gprotein per L of resin

6.3.7. Example MM 7 Demonstration of Ar Reduction with Different IonConcentrations—Adalimumab

In this example, adalimumab was chosen. Besides the conductivity rangetested presented before, lower conductivity and higher conductivityranges were also studied with the Capto Adhere. Table 61 and Table 62display the DOE study conditions using Capto Adhere columns withTris/Acetate buffer system. The load material was from Protein Aaffinity capture and pH adjusted. Column flow through pool was collectedin each run from 50 mAU of UV A280 on the ascending and 150 mAU on thedescending side of the peak. FIG. 21 demonstrates the effect of pH (6.8to 8.4), conductivity (2.3 to 13.7 mS/cm), and protein load amount (116to 354 g/L). FIG. 20 demonstrates the AR reduction at conductivity aslow as ˜1 mS/cm. Table 63 demonstrates the AR reduction at conductivity86 mS/cm with Ammonia Sulfate-Tris-Acetate buffer system.

The results demonstrated that mAb acidic species can be reduced at wideconductivity ranges from 1 to 86 mS/cm.

TABLE 61 DOE study condition Tris Acetate Edge points for Buffer RangeResponse Surface pH 7.6-8.1 7.5, 8.2 Conductivity 2.5-5.0 2.0, 5.5Protein load 150-320 116, 354 amount (g/L)

TABLE 62 DOE operting condition and results Conductivity Load amount pH(mS/cm) (g/L) ΔAR (%) Yield (%) 7.5 3.75 235 1.7 89 7.6 2.5 150 2.7 947.6 2.5 320 2.0 95 7.6 5 150 1.3 97 7.6 5 320 1.1 103 7.85 2 235 3.5 947.85 3.75 116 3.2 86 7.85 3.75 235 2.1 90 7.85 3.75 235 2.8 90 7.85 3.75354 2.2 91 7.85 5.5 235 2.1 92 8.1 2.5 150 5.0 80 8.1 2.5 320 2.6 87 8.15 150 −0.2 95 8.1 5 320 −1.1 98 8.2 3.75 235 2.9 90

TABLE 63 AR reduction and protein recovery at conductivity of 86 mS/cmand pH 7.9 Conductivity (mS/cm) pH Yield (%) ΔAR (%) 86 7.9 62 2.7 872.0 91 1.8 86 7.9 59 1.4 81 1.1 94 0.7 Note: Adalimumab in Protein Aeluate containing 25 mM acetate and 18 mM Tris or 0.89 mM Tris were pHadjusted to pH 3.5 with 3M Acetic acid solution and neutralized to pH7.9 with 3M Tris solution. One part of this viral inactivated materialwas then diluted by adding 0.3 part of a stock buffer containing 2.2M(NH₄)₂SO₄/90 mM Tris/60 mM Acetic pH 7.9 to reach conductivity of 86mS/cm.

6.3.8. Example MM 8 Demonstration of AR Reduction with Different BufferSystems with Adalimumab

In this example, molecule Adalimumab and resin Capto Adhere were chosen.The experiments were performed with different buffer systems listed inthe tables below at multiple pH and conductivity condition. The loadmaterial pH was adjusted from Protein A eluate or CEX eluate. Theresults in Table 64 and Table 65 demonstrates that mAb acidic speciescan be reduced using various buffer systems.

TABLE 64 Effect of Cation type on mAb acidic species reduction andrecovery yield Capto Adhere HEA PPA pH 7.85 pH 7.85 pH 7.85 Operating4.5 3.5 2.5 4.5 3.5 2.5 4.5 3.5 2.5 Condition mS/cm mS/cm mS/cm mS/cmmS/cm mS/cm mS/cm mS/cm mS/cm Tris/Ac % Yield 50 52 58 49 52 56 40 64 63Δ% AR 1.8 3.8 3.7 1.1 2.7 3.2 1.4 1.9 2.6 Operating ~1 mS/cm ~1 mS/cm ~1mS/cm Condition pH 8.5 pH 9.0 pH 9.5 pH 8.5 pH 9.0 pH 9.5 pH 8.5 pH 9.0pH 9.5 Arg/Ac % Yield 65 62 49 77 71 66 69 70 71 Δ% AR 8.6 6.5 1.9 4.93.5 N/R 4.5 1.9 0.6 Operating ~1 mS/cm ~1 mS/cm ~1 mS/cm Condition pH7.85 pH 8.0 pH 8.5 pH 7.85 pH 8.0 pH 8.5 pH 7.85 pH 8.0 pH 8.5 Trol/Ac %Yield 62 54 49 69 64 58 64 64 590 Δ% AR 4.1 6.0 4.6 1.7 2.9 3.0 1.4 2.12.1 Note: Load material was adalimumab from Protein A affinity captureand pH adjusted

TABLE 65 Effect of Cation type on mAb acidic species reduction andrecovery yield load amount conductivity Yield Δ % Buffer (g/L) (mS/cm)pH (%) AR Tris/Ac¹ 200 4.00 7.80 90 1.6 NaPhiosphaste/Citrate/ 200 3.537.87 87 1.5 Trolamine/NaCl² ¹Load material was adalimumab from Protein Aaffinity capture and pH adjusted ²The load material was adalimumab fromCEX capture and pH adjusted

6.3.9. Example MM 9 Demonstration of AR Reduction with Different Loading

The experiments were performed with Tris/Acetate buffer system under theconditions in table 62. The load material was adalimumab from Protein Aaffinity capture and pH adjusted. Column flow through pool was collectedin each run from 50 mAU of UV A280 on the ascending and 150 mAU on thedescending side of the peak. As seen from the profile (FIG. 22), theloading capacity has an impact on AR reduction but the AR reduction canbe achieved over a wide range of loading capacities, and is merely atrade-off between AR reduction and recovery.

6.3.10 Example MM 10 Demonstration of AR Reduction with Different LoadConcentration

In this example, Capto Adhere was chosen. The experiment was performedwith Tris/Acetate buffer system at pH 7.8±0.1 and conductivity 3.0±0.05mS/cm. The load material was adalimumab from concentrated CEX captureand pH adjusted. The prepared load material was then split to be twoparts. One was directly loaded on to a Capto adhere column; the otherpart was diluted 2 folds with equilibration buffer to make differentprotein concentration. Table 66 demonstrates that the load proteinconcentration did not have significant impact on mAb acidic speciesreduction.

TABLE 66 Adalimumab AR Reduction and Yield with Different Load ProteinConcentration Load Load Capture amount Conductivity protein Yield stepBuffer (g/L) (mS/cm) pH conc. (g/L) (%) Δ % AR CEX Tris/Ac 200 2.9 7.822.0 87 2.4 CEX Tris/Ac 200 3.0 7.7 11.0 89 2.1 CEX NaPhiosphaste/ 2003.5 7.9 4.9 87 1.5 Citrate/ Trolamine/ NaCl Protein A Tris/Ac 200 3.17.8 9.0 89 2.5 Protein A Tris/Ac 200 4.0 7.8 11.8 90 1.6 Protein ATris/Ac 200 3.0 7.8 9.9 93 2.4 Protein A Tris/Ac 208 3.0 7.8 8.4 95 3.2Protein A Tris/Ac 222 3.0 7.9 12.9 89 3.4

6.3.11. Example MM 11 Alternative Wash Modalities

In this example, molecule Adalimumab and resin Capto Adhere were chosen.The experiments were performed with Tris/acetate buffer system and theload material pH was adjusted from Protein A eluates. The equilibrationbuffer for both run was Tris/Acetic acid pH 7.8±0.1 and conductivity of3.0±0.1 mS/cm. In the gradient conductivity wash study, second bufferwas Tris/Acetic acid pH 7.8±0.1 and conductivity 6.0 mS/cm.

The results demonstrated that post load pH and conductivity can bevaried with minimal AR reduction impacted.

TABLE 67 Comparison of AR reduction and yield under different washconditions Load Load conductivity conc Yield Wash Experiment Wash(mS/cm) load pH (mg/mL) (%) CV Δ% AR Equilibration Equilibration buffer(Tris/Ac pH 3.09 7.85 9.04 89 16.4 2.5 buffer wash 7.8 and 3.0 mS/cm)wash only Gradient 1CV Equilibration buffer 3.04 7.78 7.17 91 8.0 2.2conductivity 10CV gradient conductivity wash wash from 100% Tris/Ac pH7.8, 3.0 mS/cm to 100% Tris/Ac pH 7.8, 6 mS/cm.

6.3.12. Example MM 12 Demonstration of Achievement of Absolute Value ofAR Levels in Antibody Preparations Using Mixed Mode Chromatography

In this example, molecule Adalimumab was chosen. The experiments wereperformed with multiple buffer systems and multiple MM absorbents underconditions listed in Table 68. The load materials pH was adjusted fromProtein A eluates.

The Acidic Region for Adalimumab is further grouped into two regionstermed AR1 and AR2, based on a certain retention time of the peaks seenon the WCX-10 method. The characteristics of the variants in these tworegions are expected to be different and hence the methods that reducevariants belonging to these groups can be specifically delineated.Further, in addition to achieving a certain AR reduction, it may bedesirable to achieve a certain absolute level of AR levels, inconsideration of reducing or removing certain variants. The capabilityof the current invention in achieving a certain absolute level of AR,AR1 and AR2 is demonstrated in Table 68.

TABLE 68 Acidic species level in MM resin flowthrough Conductivity YieldFT % FT % Resin Buffer pH (mS/cm) (%) AR1 AR2 Capto Adhere Tris/Ac 7.854.5 50 2.8 9.7 7.85 4.5 68 3.0 10.3 7.85 3.5 52 1.6 10.0 7.85 3.5 71 1210.5 7.85 3.0 93 3.2 9.7 7.85 2.5 58 1.7 9.4 7.85 2.5 72 2.2 10.0 Arg/Ac8.5 1 65 1.2 6.1 9.0 1 62 1.6 7.2 9.5 1 49 0.8 11.8 Trol/Ac 7.9 1 44 1.56.6 7.9 1 62 1.8 8.0 8.0 1 37 1.1 5.8 8.0 1 54 1.2 7.7 8.5 1 32 1.7 9.08.5 1 49 1.9 10.1 HEA Arg/Ac/ 8.5 1 77 1.6 8.5 9.0 1 71 0.8 12.0 PPA 8.51 69 2.2 8.7 9.0 1 70 1.0 13.5 9.5 1 71 0.7 13.1

6.3.13. Example MM 13 Demonstration of HCP and Aggregate Reduction inAddition to AR Reduction

Besides the acidic species reduction, the MM adsorbent is able to reduceother product/process related substances/impurities effectively. In theimplementation of the current invention the fact that AR reduction iseffected, other impurities/substances are expected to be clearedsignificantly as they should bind stronger than the acidic species. Thedata shown in Table 69 and Table 70 demonstrates significant HCP andaggregate reductions with different resins, buffer systems, pH,conductivities and molecules

TABLE 69 Aggregates reduction Conductivity (mS/cm) pH Buffer medium Δ %HMW D2E7 3.75 7.5 Tris/Ac Capto Adhere 0.7 2.5 7.6 Tris/Ac 0.9 2 7.85Tris/Ac 0.9 3.75 7.85 Tris/Ac 1.0 5.5 7.85 Tris/Ac 0.7 2.5 8.1 Tris/Ac1.0 3.75 8.2 Tris/Ac 0.8 4.0 8.2 Tris/Ac 1.0 8.0 6.8 Tris/Ac 0.2 8.0 8.4Tris/Ac 1.0 1.0 8.5 Arg/Ac Capto Adhere 0.5 1.0 9.0 Arg/Ac 0.8 1.0 9.5Arg/Ac 0.9 1.0 8.5 Arg/Ac HEA 0.4 1.0 9.0 Arg/Ac 2.5 1.0 9.5 Arg/Ac 0.71.0 8.5 Arg/Ac PPA 0.5 1.0 9.0 Arg/Ac 2.8 1.0 9.5 Arg/Ac 0.4 MAB C 3.0 8Tris/Ac Capto Adhere 1.0 3.0 8.5 Tris/Ac Capto Adhere 1.1 3.0 9 Tris/AcCapto Adhere 0.6 3.0 8 Tris/Ac PPA 0.7 3.0 8.5 Tris/Ac PPA 0.5 3.0 8Tris/Ac HEA 0.7 3.0 8.5 Tris/Ac HEA 0.6

TABLE 70 HCP Log reduction Condutivity (mS/cm) pH Buffer medium HCP LRFD2E7 3.75 7.5 Tris/Ac Capto Adhere 1.5 2.5 7.6 Tris/Ac 1.7 2.0 7.85Tris/Ac 2.2 3.75 7.85 Tris/Ac 1.9 5.5 7.85 Tris/Ac 1.4 2.5 8.1 Tris/Ac2.3 3.75 8.2 Tris/Ac 2.1 4.0 8.2 Tris/Ac 1.7 8.0 6.8 Tris/Ac 0.3 8.0 8.4Tris/Ac 0.7 MAB B 3 6.8 Tris/Ac Capto Adhere 2.0 4.5 6.8 Tris/Ac CaptoAdhere 1.3 3 6.8 Tris/Ac PPA 1.2 4.5 6.8 Tris/Ac 1.2 3 6.8 Tris/Ac HEA1.3 4.5 6.8 Tris/Ac 1.1

6.3.14. Example MM 14 Combinations of MM with Alternative SeparationStrategies

Acidic Species Reduction by MM Adsorbents is expected to be performedafter capture of the antibody by other means, or after one or moreintermediate steps following the capture step. In the examples below theMM Adsorbent steps were performed either following a Cation ExchangeCapture step or Protein A affinity capture step. As shown in Table 71,AR reduction was achieved at two different conductivities followingProtein A Chromatography and CEX Chromatography.

TABLE 71 AR Reduction with different source materials Capture Bufferconductivity (mS/cm) pH Yield (%) Δ % AR Protein A Tris/Ac 3.1 7.8 892.5 Protein A Tris/Ac 4.0 7.8 90 1.6 CEX Tris/Ac 2.9 7.8 87 2.4 CEXTris/Ac 3.0 7.7 89 2.1

Adalimumab was purified by a CEX chromatography step followed with a lowpH viral inactivation step. The filtered viral inactivated material wasbuffer exchanged and loaded onto a Capto Adhere column. The flowthroughof Capto Adhere material was then purified with a HIC column withbind/elute mode. As shown in Table 72, AR reduction was achievedprimarily with MM step, with some contribution from other steps.

TABLE 72 Complete Process train with CEX Chromatography Capture- ARReduction Δ % AR Δ % Lys Yield (%) CEX eluate n/a n/a n/a MM Load 0.290.34 90% MM Flowthrough 2.57 2.57 93% HIC eluate 0.95 0.94 97%

Adalimumab was purified by a Protein A chromatography step followed witha low pH viral inactivation step. The filtered viral inactivatedmaterial was buffer exchanged and loaded onto a Capto Adhere column. Theflowthrough of Capto Adhere material was then purified with a HIC columnwith bind/elute mode as well as Flow Through mode. As shown in Table 73,AR reduction was achieved primarily with MM step, with some contributionfrom other steps.

TABLE 73 Complete Process Train with Protein A Capture - AR, HMW and HCPreduction Yield % AR % HMW Process (%) reduction reduction HCP LRFClarified Harvest 97.0% n/a n/a n/a Prt-A Eluate Pool 89.6% 0.06 1.87Viral Inactivated 99.7% No reduction 0.07 0.39 Filtrate MM FT pool 91.9%2.26 0.83 1.63 HIC (B/E) Eluate 90.1% 0.40 0.22 1.41 NanofiltrateFiltrate 90.7% No reduction No reduction 0.15 BDS (B/E) 102.0% Noreduction No reduction 0.22 HIC FT-pool 98.5% 0.16 0.23 0.46 VF(FT)Filtrate 96.1% No reduction No reduction 0.10 BDS (FT) 103.8% Noreduction No reduction No reduction

6.3.15. Example MM 15 Utility of AR Reduction

The current invention provides a method for reducing acidic species fora given protein of interest. In this example adalimumab was preparedusing a combination of AEX and CEX technologies to produce a Low-AR andHigh-AR sample with a final AR of 2.5% and 6.9%, respectively. Bothsamples were incubated in a controlled environment at 25° C. and 65%relative humidity for 10 weeks, and the AR measured every two weeks.FIG. 23 shows the growth of AR for each sample over the 10 weekincubation. It is evident from FIG. 23 the growth rate of AR is linearand similar between both the Low-AR and High-AR samples. Based on theseresults the reduced AR material can be stored 3 fold longer beforereaching the same AR level as the High-AR sample. This is a significantutility as this can be very beneficial in storage handling and use ofthe antibody or other proteins for therapeutic use.

6.4 Process Combinations to Achieve Target % Ar or Ar Reductions

Upstream and Downstream process technologies, e.g., cell culture andchromatographic separations, of the inventions disclosed in thefollowing applications can be combined together or combined with methodsin the art to provide a final target AR value or achieve a % ARreduction, as well as to, in certain embodiments, reduce product relatedsubstances and/or process related impurities. Upstream methods for ARreduction include, but are not limited to those described in the in theU.S. patent application having attorney reference no. 082254.0235.Downstream methods for AR reduction include, but are not limited to,those described in the instant application. Exemplary technologiesdisclosed in the referenced applications include, but are not limitedto: cell culture additives & conditions; clarified harvest additives andpH/salt conditions; mixed mode media separations; anion exchange mediaseparations; and cation Exchange media separations.

The instant example demonstrates the combined effect of one or more ofthese technologies in achieving a target AR value or AR reduction,thereby facilitating the preparation of an antibody material having aspecific charge heterogeneity. Additional examples of combinations ofdownstream technologies and upstream technologies are provided in thereferenced applications.

In this example, the combination of upstream and downstream methodsinvolves the reduction of acidic species in 3 L bioreactor cell culturessupplemented with arginine (2 g/l) and lysine (4 g/l) as has beenpreviously demonstrated in the U.S. patent application having attorneyreference no. 082254.0235. The results of that strategy are summarizedin Table 74. The total acidic species was reduced from 20.5% in thecontrol sample to 10.2% in sample from cultures that were supplementedwith the additives. In this study, Adalimumab producing cell line 1 wascultured in media 1 (chemically defined media) supplemented with aminoacid arginine (2 g/l) and lysine (4 g/l) in a 300 L bioreactor. On Day12 of culture, the culture was harvested and then subsequently analyzedusing WCX-10 post protein A purification and the percentages of totalpeak(s) area corresponding to the acidic species were quantified. Thepercentage of acidic species was estimated to be 9.1% in the 300 Lharvest sample.

TABLE 74 AR levels achieved with use of upstream technologies 3 LBioreactor 300 L Bioreactor Arginine (2 g/l) + Arginine (2 g/l) +Control Lysine (4 g/l) Lysine (4 g/l) Total Total Total AR1 AR2 AR AR1AR2 AR AR1 AR2 AR (%) (%) (%) (%) (%) (%) (%) (%) (%) 6.3 14.2 20.5 2.67.6 10.2 2.4 6.7 9.1

The material produced by the 300 L Bioreactor employing Arginine andLysine additions, that effectively reduced the AR levels to 9.1% waspurified using a downstream process employing Mixed Mode chromatographyas the primary AR Reduction method.

Adalimumab was purified by a Protein A chromatography step followed witha low pH viral inactivation step. The filtered viral inactivatedmaterial was buffer exchanged and loaded onto a Capto Adhere column. Theflowthrough of Capto Adhere material was then purified with a HIC columnwith bind/elute mode as well as Flow Through mode. As shown in Table 75,AR reduction was achieved primarily with MM step, with some contributionfrom other steps. The table also shows that additional product relatedsubstances such as aggregates and process related impurities such as HCPcan be effectively reduced employing these combined technologies.

TABLE 75 Complete Downstream Process Train with Protein A Capture - AR,HMW and HCP reduction Yield % AR % HMW Process (%) reduction reductionHCP LRF Clarified Harvest 97.0% n/a n/a n/a Prt-A Eluate Pool 89.6% 0.061.87 Viral Inactivated 99.7% No reduction 0.07 0.39 Filtrate MM FT pool91.9% 2.26 0.83 1.63 HIC (B/E) Eluate 90.1% 0.40 0.22 1.41 NanofiltrateFiltrate 90.7% No reduction No reduction 0.15 BDS (B/E) 102.0% Noreduction No reduction 0.22 HIC FT-pool 98.5% 0.16 0.23 0.46 VF(FT)Filtrate 96.1% No reduction No reduction 0.10 BDS (FT) 103.8% Noreduction No reduction No reduction

As is evident from the above example, the MM method further reduced theAR levels, by 2.26%. Therefore upstream technologies for reduction canbe combined with downstream technologies to achieve AR levels/ARreduction.

Patents, patent applications, publications, product descriptions,GenBank Accession Numbers, and protocols that may be cited throughoutthis application, the disclosures of which are incorporated herein byreference in their entireties for all purposes. For example, but not byway of limitation, patent applications designated by the followingattorney docket numbers are incorporated herein by reference in theirentireties for all purposes: 082254.0104; 082254.0235; 082254.0238;082254.0242; and 082254.0243.

What is claimed is:
 1. A method for reducing process-related impuritiesand/or product-related substances in a preparative scale sample of aprotein of interest comprising at least one process-related impuritiesand/or product-related substance, the method comprising: (a) contactingthe sample to a chromatography media, wherein the contact occurs in thecontext of a loading buffer; (b) washing the chromatography media with awash buffer that is the same, or substantially the same as the loadingbuffer; and (c) collecting a chromatography sample, wherein thechromatography sample comprises a protein of interest and a reducedamount of process-related impurities and/or product-related substances.2. The method of claim 1, wherein the chromatography media is selectedfrom the group consisting of anion exchange, cation exchange, and mixedmode media.
 3. The method of claim 1, wherein the chromatography mediais a mixed mode adsorbent material comprising cation exchange andhydrophobic interaction functional groups.
 4. The method of claim 3,wherein the mixed mode resin is Capto MMC resin.
 5. The method of claim2, wherein the cation exchange (CEX) adsorbent material is selected fromthe group consisting of a CEX resin and a CEX membrane adsorber.
 6. Themethod of claim 5, wherein the CEX resin is the Poros XS resin.
 7. Themethod of claim 1, wherein the pH of the loading and wash buffers arelower than the isoelectric point of the protein of interest.
 8. Themethod of claim 1, wherein the chromatography sample contains a reducedlevel of acidic species.
 9. The method of claim 1, wherein thechromatography sample contains a reduced level of protein fragments. 10.The method of claim 1, wherein the chromatographic samples contains areduced level of host cell proteins.
 11. The method of claim 1, whereinthe chromatographic sample contains levels of basic variants that differfrom the starting load material.
 12. The method of claim 1, wherein theprotein of interest is an anti-TNFα antibody.
 13. A pharmaceuticalcomposition comprising a process-related impurity-reduced and/orproduct-related substance-reduced composition comprising a protein ofinterest and a pharmaceutically acceptable carrier.
 14. Thepharmaceutical composition of claim 14, wherein said composition issubstantially free of product-related aggregates, product-relatedfragments, and HCPs.